Methods for expressing proteins in axons
The invention relates to expressing proteins in the axons of mammalian neurons. The invention provides nucleic acids that can be used to express a selected polypeptide in neuronal axons, viruses that can be used deliver nucleic acids of the invention into neuronal axons, as well as methods for doing so. Thus, the invention provides pharmaceutical compositions comprising viruses of the invention, as well as their use in methods of treating injured axons or conditions associated with aberrant axon growth or function.
1. A method for translating a polypeptide in the axon of a mammalian neuron comprising applying a recombinant alphavirus exclusively to the axon of the neuron, wherein the recombinant alphavirus is capable of infecting the axon of a mammalian neuron, wherein the alphavirus comprises a single-stranded RNA comprising:
(a) a mammalian translation initiation element comprising a 5′CAP or an internal ribosome entry site (IRES); and
(b) a polypeptide coding sequence operably-linked to the mammalian translation initiation element, wherein the coding sequence of the single-stranded RNA is locally translated within the axon to generate a polypeptide within the axon of a mammalian neuron, and the polypeptide modulates the growth or function of an axon.
2. The method of claim 1 , wherein the polypeptide promotes the growth of an injured axon.
3. The method of claim 1 , wherein the polypeptide reduces the activity the axon of a neuron.
4. The method of claim 1 , wherein the polypeptide is a kinase or a transcription factor.
5. The method of claim 4 , wherein the kinase is a src kinase.
6. The method of claim 4 , wherein the transcription factor is a cyclic AMP-response element-binding protein (CREB) or nervy.
7. The method of claim 1 , wherein the polypeptide is C3-ADP-ribosyltransferase, a dominant-negative RhoA mutant polypeptide, a cAMP-producing enzyme, glutamic acid decarboxylase, human proenkephalin, an inhibitor of a dominant-negative Vps24, an intestinal peptide (VIP), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial-derived neurotropic factor (GDNF), GAP 43, CAP23, a myc-tagged soluble adenylyl cyclase, a green fluorescent protein (GFP), a myristoylated GFP, a destabilized enhanced GFP (dEGFP), a myristoylated dEGFP, Cherry, or a myc-tagged Cherry.
8. The method of claim 7 , wherein the dominant-negative RhoA mutant polypeptide is N19-RhoA polypeptide.
9. The method of claim 7 , wherein the cAMP-producing enzyme is a soluble adenylyl cyclase.
10. The method of claim 1 , further comprising locally applying to the axon a brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial-derived neurotropic factor (GDNF), nerve growth factor or a combination thereof.
11. The method of claim 1 , wherein the axon is that of a sensory neuron, an upper motor neuron or a dorsal root ganglion.
12. The method of claim 11 , wherein the sensory neuron is a peripheral sensory neuron.
13. The method of claim 1 , wherein the alphavirus is locally applied to the axon at a site of injury.
14. The method of claim 1 , wherein the virus is an attenuated form of the alphavirus.
15. The method of claim 14 , wherein the alphavirus is a Sindbis virus or a Semliki forest virus.
16. The method of claim 1 , Wherein the nucleic acid further comprises a poly-adenylyl tail.
17. The method of claim 1 , wherein the IRES is a eukaryotic sequence.
18. The method of claim 1 , wherein the IRES is an encephalomyocarditis sequence, or a Semliki forest viral sequence.
19. The method of claim 1 , wherein the IRES comprises the sequence of any one of SEQ ID NO: 29-35.
20. The method of claim 1 , wherein the mammalian translation initiation element is an internal ribosome entry site (IRES) that replaces a subgenomic promoter in the isolated nucleic acid.