AAV-mediated subcellular targeting of heterologous rhodopsins in retinal ganglion cells

A recombinant adeno-associated virus-2 (rAAV2) expression vector comprising a nucleic acid molecule encoding a rhodopsin for expression in subcellular regions of a retinal neuron comprising:

• (a) a first nucleotide sequence encoding a light-gated channel rhodopsin or a light-driven ion pump rhodopsin;
• (b) linked in frame to (a), a second nucleotide sequence encoding a peptide or polypeptide sorting motif, wherein the second nucleotide sequence is selected from the group consisting of:
• (1) a nucleotide sequence encoding nicotinic acetylcholine receptor a7 subunit (nAchR) comprising SEQ ID NO:
• 9, (2) a nucleotide sequence encoding voltage-gated potassium channel 4.2 (Kv4.2) comprising SEQ ID NO:
• 11, (3) a nucleotide sequence encoding telencephalin (TLCN) comprising SEQ ID NO:
• 13, and (4) a nucleotide sequence encoding AMPA receptor GluR1 subunit comprising SEQ ID NO:
• 15:
• (c) operatively linked to (a) and (b), a promoter sequence;
• and
• (d) a polyadenylation sequence,
• wherein the nucleic acid molecule is flanked at its 5′ end by a 5′ inverted terminal repeat (ITR) of AAV2 and at its 3′ end by a 3′ ITR of AAV2, wherein a nucleotide sequence of the 5′-ITR is set forth in SEQ ID NO:
• 17, and wherein a nucleotide sequence of the 3′-ITR is set forth in SEQ ID NO:
• 18.

A recombinant adeno-associated virus-2 (rAAV2) expression vector comprising a schematic structure selected from:

• (a) 5′-ITR-CAG-ChR2-GFP-{Motif}-WPRE-bGHpoly A-ITR-3′,
• (b) 5′-ITR-CAG-ChR2-{Motif}-WPRE-bGHpoly A-ITR-3′,
• (c) 5′-ITR-CAG-HalOR-GFP-{Motif}-WPRE-bGHpolyA-ITR-3′, and
• (d) 5′-ITR-CAG-HaloR-{Motif}-WPRE-bGHpoly A-ITR-3′;
• wherein:
• ITR is a AAV2 inverted terminal repeat,
• CAG is a cytomegalovirus enhancer/chicken β-actin promoter,
• ChR2 is a nucleotide sequence encoding channelrhodopsin-2,
• GFP is a nucleotide sequence encoding a green fluorescent protein,
• Motif is a nucleotide sequence encoding a sorting motif selected from the group consisting of:
• (1) a nucleotide sequence encoding nicotinic acetylcholine receptor a7 subunit (nAchR) comprising SEQ ID NO:9,
• (2) a nucleotide sequence encoding voltage-gated potassium channel 4.2 (Kv4.2) comprising SEQ ID NO:11,
• (3) a nucleotide sequence encoding telencephalin (TLCN) comprising SEQ ID NO:
• 13, and
• (4) a nucleotide sequence encoding AMPA receptor GluR1 subunit comprising SEQ ID NO:
• 15,
• WPRE is a woodchuck hepatitis virus posttranscriptional regulatory element,
• bGHpolyA is a polyA sequence from bovine growth hormone,
• and
• wherein any two or more of the ChR2, GFP, and Motif, or of the HaloR, GFP, and Motif, are linked in frame.