Compositions and methods for targeted ubiquitination of the voltage-gated sodium channel nav1.8

Abstract

Compositions and methods for targeted ubiquitination of the voltage-gated sodium channel Nav1.8. The compositions include a bifunctional molecule that includes two bioactive domains, a first bioactive domain for selective binding to Nav1.8 channels and a second bioactive domain for which can be has the activity of (i) catalyzing a ubiquitination reaction to tag the channel for internalization and degradation, (ii) recruitment and binding of a ubiquitin ligase which then catalyzes a ubiquitination reaction to tag the channel for internalization and degradation or (iii) l recruitment of the fusion protein to lysosomes. The bifunctional molecule includes a HECT domain from NEDD4 ubiquitin ligase and includes a myosin tail domain (MTD) from clathrin linker-1 (SCLT-1), functional fragments or variants thereof, provide the two bioactive domains. The compositions are administered to a subject in need thereof, in an effective amount to treat acute chronic pain.

Description

[0001]ATTORNEY DOCKET NO. YU 8723 PCT COMPOSITIONS AND METHODS FOR TARGETED UBIQUITINATION OF THE VOLTAGE-GATED SODIUM CHANNEL NAV1.8 CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 580,094 filed September 1, 2023, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under T32GM007205 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION The disclosed invention is generally in the field of pain management and specifically in the area of compositions and method for treating pain. BACKGROUND OF THE INVENTION Over one-quarter of US citizens suffer from chronic pain. The scourge of chronic pain is devastating in both its impact on our nation’s health and its financial implications. In aggregate, the United States spends over 600 billion dollars per year on costs associated with the pain epidemic - exceeding that of cancer, diabetes, and heart disease combined2. Despite an incredible demand, therapeutic options for chronic pain suffer from severe limitations. Opioids are a mainstay of treatment for many types of pain, but they are ineffective in chronic pain and their use is associated with multiple side effects including physical dependency and opioid use disorder3. Other classes of pain-management medications (NSAIDs, etc.) have only mild-to- moderate potency and are also linked to a slew of adverse effects4. By studying the neurological basis of pain, points along the pain pathway can be identified which may be susceptible to pharmacologic therapy. Human pain sensation begins with sodium flux through peripheral voltage-gated sodium (Nav) channels5. The role of NaVchannels in human pain transmission is evident in the action of local anesthetic agents like lidocaine, which non selectively block NaVchannels along the length of the primary sensory axon and result in the complete block of action potential propagation6. Nav channels are composed of α-subunits, which are the pore-forming and voltage-sensitive machinery of the channel and auxiliary α subunits as well as other multiple channel partners that can regulate their gating properties, trafficking and distribution7. There are 9 α-subunit isoforms in humans (NaV1.1-1.9), each with a pattern of tissue-specific expression8. NaV1.7, NaV1.8, and NaV1.9 are the principal ion channels responsible for action potential electrogenesis and propagation in peripheral nociceptors in adults5,9. NaV1.7 and NaV1.9 are responsible for the amplification of sub-threshold depolarizations, while NaV1.8 is the main contributor to the rising phase of the45678892.11 ATTORNEY DOCKET NO. YU 8723 PCT nociceptive action potential and the total loss of this channel abrogates repetitive firing in DRG neurons5,9. A role for Nav1.8 in human pain disorders is supported by the findings that gain-of- function mutations in NaV1.8 result in painful neuropathies16, and a moderate loss-of-function mutation in NaV1.8 has recently been associated with reduced pain sensitivity17. To date, the principal strategy for targeting these channels has been to directly inhibit sodium conductance through the channel18. While promising, agents with this mechanism of action have not yet been deployed in the clinic. Thus, more effective and non-addictive treatments for pain are urgently needed for pain management, especially chronic pain. An object of the invention is to provide compositions for treating pain in a subject. Another object of the invention is to provide methods of treating pain in a subject in need thereof. SUMMARY OF THE INVENTION Compositions for treating pain and methods of use thereof for pain management, are provided. The compositions include a fusion protein (herein, pain active agents) or nucleic acids encoding the fusion protein. In some forms the fusion protein includes a bioactive module for catalyzing a ubiquitination reaction (this fusion protein referred to herein as “UbiquiNav”), or an inducible UbiquiNav (described below). In some forms, the fusion protein includes module recruiting / binding an endogenous ubiquitin ligase i.e., a ubiquitin ligase recruiting / binding domain (herein, R-UbiquiNav or iR-UbiquiNAv, described below). In some forms the fusion protein includes a bioactive module for lysosomal targeting / recruiting of the fusion to lysosome (LR-Nav or iLR-Nav). UbiquiNav, R- UbiquiNav, LR-Nav, iUbiquiNav, iR- UbiquiNav, iLR-Nav are referred to, collectively as “pain active agents”, and individually as “pain active agent”. UbiquiNav is a bifunctional molecule that includes two bioactive modules, a first bioactive module for selective binding to Nav1.8 channels and a second bioactive module for catalyzing a ubiquitination reaction to tag the channel for internalization and degradation. R- UbiquiNav is a bifunctional molecule that includes two bioactive modules, a first bioactive module for selective binding to Nav1.8 channels and a second bioactive module for recruiting the binding of an endogenous ubiquitin ligase, which catalyzes a ubiquitination reaction to tag the channel for internalization and degradation. LR-Nav is a bifunctional molecule that includes two bioactive modules, a first bioactive module for selective binding to Nav1.8 channels and a second bioactive module for recruiting the fusion protein to lysosomes.45678892.12 ATTORNEY DOCKET NO. YU 8723 PCT In some forms, the first and second bioactive modules are separated by one or more flexible linkers. UbiquiNav, R- UbiquiNav or LR-Nav can be represented by the general formula I D1 / D2-L1-D2 / D1, where D1 is a first bioactive module (for example, for selective binding to Nav1.8 channels), D2 is a second bioactive module (for example, for catalyzing a ubiquitination reaction), and L1 is an optional flexible linker, for example, an amino acid linker. In some forms, D1 / D2 includes an MTD (myosin tail domain) sequence preferably, from clathrin linker-1 (SCLT-1, previously called clathrin-associated protein 1A, CAP-1A) for UbiquiNav , R- UbiquiNav or LR-Nav. D1 / D2 and D2 / D1 as shown in Formula I means the bioactive domain that is selective for binding to Nav1.8 channels can be in the N or C- terminal of the fusion protein, and the second bioactive domain also be in the N or C-terminal of the fusion protein. For UbiquiNav, D1 / D2 includes the catalytic domain from a ubiquitin ligase, such as the RING (Really Interesting New Gene), or the HECT (homologous to the E6-AP C-terminus) sequence from NEDD4 ubiquitin ligase, preferably, NEDD4-2. In some forms, D1 includes the HECT sequence from NEDD4ubiquitin ligase and D2 includes an MTD sequence. The MTD sequence and the HECT sequence is from NEDD4ubiquitin ligase are preferably, human NEDD4 -2 (also known as NEDD4-L)( hNedd4L => NM_001144967) . For R-UbuquiNav, the compositions include a recruiting sequence for a ligase, such the ligase Kelch-like ECH-associated protein 1 (KEAP1) or a functional fragment or variant thereof, as D1 / D2. In some forms, for R-UbuquiNav, the composition incorporates a degron, as D1 / D2. For LR-Nav, the composition incorporates a lysosome recruiting sequence, as D1 / D2. In some embodiments, the UbiquiNav , R-UbiquiNav or LR-Nav fusion polypeptide can further include a functional element such as a protein transduction domain, fusogenic polypeptide, targeting signal, or expression and / or purification tag. Thus, the UbiquiNav, R-UbiquiNav or LR-Nav fusion polypeptide can be a fusion protein including a UbiquiNav, R-UbiquiNav or LR- Nav fusion protein sequence or fragment or variant thereof, and a heterologous sequence. In some forms, D1 or D2 can be SEQ ID NO:2 (a HECT catalytic sequence). or polypeptide variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 3. In some forms, D1 / D2 can be SEQ ID NO: 3 (a HECT catalytic sequence) or polypeptide variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3.45678892.13 ATTORNEY DOCKET NO. YU 8723 PCT In some forms, D1 or D2 can be SEQ ID NO:26 (a KEAP1 recruitment sequence) or polypeptide variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:26.In some forms, D1 or D2 can be SEQ ID NO:27 (a degron) or polypeptide variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:27. In some forms, rather than a domain for a ubiquitin ligase, the composition incorporates a lysosome recruitment peptide domain which brings Nav1.8 directly to the lysosome by leveraging the action of one or more non-ubiquitin proteins. An exemplary lysosome recruitment domain brings Nav1.8 to the lysosome directly by leveraging the action of the protein LAMP2A (lysosomal associated membrane protein 2). Another lysosome recruitment domain brings Nav1.8 directly to the lysosome by leveraging the action of the proteins hsc70 (heat shock protein 70) and LAMP2A. In some forms, D1 or D2 can be SEQ ID NO:28 (a lysomal recruitment sequence) or polypeptide variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:28. In some forms, D1 or D2 can be SEQ ID NO:29 (a lysomal recruitment sequence) or variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29. In some forms, D1 or D2 can be SEQ ID NO:35 (a lysomal recruitment sequence) or variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:35. In some forms, D1 / D2 can be SEQ ID NO:5 (an MTD sequence) or variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5. In some forms, the fusion protein is i UbiquiNav, iR- UbiquiNav or iLR-Nav produced from a fusion protein represented by the general formula: D’1-P1-L1-D’2-P2, Where, D’1 is a first bioactive module; D’2 is a second bioactive module, L1 is an optional flexible linker, for example, an amino acid linker,45678892.14 ATTORNEY DOCKET NO. YU 8723 PCT P1 is a first peptide binding domains of the anti-fungal small molecule Mandipropamid, and P2 is a second peptide binding domains of the anti-fungal small molecule Mandipropamid. Alternatives for D’1 and D’2 are the same as disclosed herein for D1 and D2 in UbiquiNav, R- UbiquiNav or LR-Nav, for iUbiquiNav, iR- UbiquiNav and iLR-Nav, respectively. L1 alternatives are the same as disclosed herein for UbiquiNav. In iUbiquiNav, for example, the two critical modules of UbiquiNav (MTD – selective binding; HECT of NEDD4L – catalytic) are linked to orthogonal binding proteins (PYR1 (Pyrabactin Resistance 1), ABI1 (Abscicic Acid Receptor)) that recognize different sites of Mandipropamid, a small molecule (FIG.2B). Transfection of neurons with a cDNA that encodes both PYR1-MTD and ABI1-HECT on the same mRNA transcript separated by a peptide cleavage motifs allows equal stoichiometries of the two halves of iUbiquiNav to be expressed in sensory neurons. In the absence of Mandipropamid, these constructs remain separate (though the PYR1-MTD should still bind to the C-terminus of NaV1.8. In the presence of Mandipropamid, the PYR1-MTD and ABI1-HECT modules should rapidly dimerize, yielding a functional UbiquiNav fusion protein that then ubiquitinates NaV1.8. Thus, iUbiquiNav is two separate fusion proteins of D’1-P1 and D’2-P2, which are rendered active by coadministration of an agent that dimerizes P1 and P2. The same design is disclosed herein for iR- UbiquiNav or iLR-Nav, where HECT is replaced with the ubiquiting ligase recruiting sequence or the lysosomal recruiting sequence. In some forms L1 is an amino acid sequence such as GSTG (SEQ ID NO: 6); Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:7), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO:8), (Gly4-Ser)3 (SEQ ID NO:9), (Gly4-Ser)4 (SEQ ID NO:10), and GSGSGSGS (SEQ ID NO:11). Additional peptides include GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:12 which can be included in the construct for iUbiquiNav to direct the production of two independent proteins from the same mRNA. Nucleic acids encoding UbiquiNav, R- UbiquiNav, or LR-Nav polypeptides are also provided. The nucleic acids can be, for example, RNA or DNA, and optionally include an expression control sequence(s) such as promoter, which may be cell type specific (e.g., nerve cells). In some embodiments, the nucleic acid is a vector such as a plasmid or viral vector, or an mRNA. In some embodiments, the nucleic acid has one or more functional elements such as a protein transduction domain, fusogenic polypeptide, and / or targeting signal conjugated thereto. Any of the disclosed polypeptides and nucleic acids can be packaged in or otherwise associated with a delivery vehicle. Exemplary delivery vehicles are also provided and can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, or45678892.15 ATTORNEY DOCKET NO. YU 8723 PCT multilamellar vesicles. Optionally, the delivery vehicles have one or more of a protein transduction domain, fusogenic polypeptide, and / or targeting signal conjugated thereto. Pharmaceutical compositions are also provided and can include a pharmaceutically acceptable carrier and any of the disclosed polypeptides or nucleic acids alone or packaged in a delivery vehicle. Methods of treatment are provided and typically include administering a subject in need thereof an effective amount of a disclosed fusion polypeptide or nucleic acid, optionally, but preferably, in a pharmaceutical composition. The methods include delivering / administering an effective amount of the pain active agentto the subject. In some forms the methods include delivering UbiquiNav in the form of a polypeptide. In some forms, the methods include delivering a vector encoding the pain active agent, to tissue / cells in the subject, such as sensory neurons. In some forms, delivering the pain active agent to tissues in living animals depends on introducing genetic material encoding the pain active agentto peripheral sensory neurons. One approach to this delivery is expressing UbiquiNav in adeno associated virus (AAV) capsid which can be delivered intrathecally to reach the tissues where sensory neurons reside. Data in this application demonstrates that UbiquiNav cloned into an AAV shuttle plasmid, driven by the active promoter CMV, produces suppresses of the Nav1.8 channels when delivered in vivo. Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions. FIGs.1A-1I shows the development of UbiquiNaV, a heterobifunctional degrader of NaV1.8 channels. FIG.1A is a schematic illustrating the proposed strategy of targeting NaV1.8 by degrading existing sodium channels at the cell surface. To degrade a membrane protein, two critical modules are required. (1) A sequence-selective binding module and (2) A catalytic module that triggers ubiquitination of the substrate. Ubiquitination of the target results in internalization and degradation by the proteasome. FIG.1B shows overexpression of Sclt145678892.16 ATTORNEY DOCKET NO. YU 8723 PCT reduces endogenous NaV1.8 current amplitude and density in rat pup DRG neurons. Whole-cell voltage clamp recordings of DRG neurons from 2-4 d old rat pups transfected with plasmids encoding Sclt1-P2A-eGFP or eGFP. The amino acid sequence of P2A is: SRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 24). The inset shows the recording protocol of endogenous TTX-R NaV1.8 currents in the presence of TTX to block endogenous TTX-S currents. Note that sodium currents through voltage-gated sodium channels are inward (sodium flows from outside of the cell to the inside). Inward current is defined as negative by convention. Inward current is defined as negative by convention. FIG.1C is a bar graph showing the inward peak Na+ current density through endogenous NaV1.8 channels is significantly reduced in neurons expressing Sclt1-2A-eGFP (first bar; n=6), compared to eGFP- control (2ndbar; n=5). FIG.1D is a bar graph showing inward peak Na+ current through endogenous NaV1.8 channels is significantly reduced in neurons expressing Sclt1-2A-eGFP (1stbar; n=6), compared to eGFP-control (2ndbar; n=5). FIG.1E shows that expression of the MTD of Sclt1 alone does not affect endogenous Nav1.8 current amplitude and density in rat pup DRG neurons. Whole-cell voltage clamp recordings of DRG neurons from 2-4 d old rat pups transfected with plasmids encoding mRuby2-P2A-MTD or mRuby2-control. FIG.1F is a bar graph showing inward peak Na+ current density through endogenous NaV1.8 channels in neurons transfected with mRuby2-P2A-MTD (1stbar; n=9) or mRuby2-control (2ndbar; n=11). FIG.1G is a bar graph showing the inward peak Na+ current through endogenous NaV1.8 in neurons transfected with mRuby2-P2A-MTD (1stbar; n=9) or mRuby2-control (2ndbar; n=11). FIG.1H shows overexpression of rat Sclt1 reduces human Nav1.8 current amplitude and density. DRG neurons from Nav1.8-null adult mice were co-transfected with mCherry-Nav1.8 and either Sclt1-P2A-eGFP (Sclt1) or eGFP (control). Inward currents were assessed by whole- cell voltage clamp. FIG.1I (top panel) shows a schematic of UbiquiNav. UbiquiNav is composed of two modules separated by a linker. The Nav1.8 binding module is the MTD of Sclt1, and the ubiquitinating catalytic module is the HECT domain of NEDD4L. An eGFP reporter is fused in frame a P2A linker. Bars: Mean ± SEM. ns p> 0.05, ** p<0.01 by the Mann- Whitney U-test. The bottom panel shows detection of UbiquiNav at steady state levels in HEK293 cells, indicating the construct does not self-degrade. FIGs.2A-2K shows that UbiquiNav decreases Nav1.8 current density and neuronal surface expression. FIG.2A shows the effect of UbiquiNav on NaV1.8 current density in rat pup DRG neurons, and identify that this effect is dependent on the catalytic activity of the HECT domain. Rat pup DRG neurons were transfected with plasmids encoding UbiquiNav (control), eGFP, or catalytically inactivated UbiquiNav (UbiquiNav-inact). Nav1.8 current densities were45678892.17 ATTORNEY DOCKET NO. YU 8723 PCT evaluated by whole-cell voltage-clamp recordings. FIG.2B shows whole-cell voltage clamp recordings of DRG neurons from 2-4 d old rat pups transfected with plasmids encoding UbiquiNaV, UbiquiNaV C942S, or eGFP-control. FIG.2C is a bar graph showing the inward peak Na+ current density through NaV1.8 in neurons transfected with UbiquiNaV (1stbar; n=30), UbiquiNaV C942S (2ndbar; n=14), or eGFP-control (3rdbar; n=21). FIG.2D is a schematic depicting the membrane topology of Halo-NaV1.8, which contains an extracellular HaloTag enzyme. FIG.2E is a schematic of surface labeling experiments. Cell-impermeant JF635i- HaloTag is added to the somatic and axonal chambers of MFCs containing DRG neurons expressing Halo-NaV1.8-P2A-mRuby2 and either eGFP-control or UbiquiNaV. Following labeling, neurons are fixed with 4% PFA and imaged with confocal microscopy. FIG.2F is a bar graph showing quantification of somatic Halo-NaV1.8 surface expression in neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP-control (1stbar; n=25) or UbiquiNaV (2ndbar; n=44). FIG.2G is a bar graph showing quantification of axonal Halo-NaV1.8 surface expression in neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP-control (1stbar; n=29) or UbiquiNaV (2ndbar; n=49). FIG.2H is a schematic of OPAL imaging experiments to evaluate anterograde trafficking of NaV1.8. Cell permeable JFX650-HaloTag ligand is added to the somatic chamber of MFCs containing DRG neurons expressing Halo-NaV1.8-P2A-mRuby2 and either eGFP-control or UbiquiNaV. Following ligand incubation in the somatic chamber, the axonal chamber is imaged by confocal microscopy to reveal anterogradely traveling vesicles carrying labeled Halo-NaV1.8. FIG.2I is a bar graph showing quantification of anterograde vesicular flux of Halo-NaV1.8 channels in neurons transfected with eGFP-control (first bar; n=25) or UbiquiNaV (second bar; n=25). Bars: Mean ± SEM. ns p> 0.05, * p<0.05, ** p<0.01, **** p<0.0001 by the Mann-Whitney U-test. Multiple comparisons corrected by quantifying the False Discovery Rate and adjusting p-values. FIG.2J is a schematic of the chemically induced dimerization approach of iUbiquiNav. The two critical domains of UbiquiNav (MTD – selective binding; HECT of NEDD4L – catalytic) are linked to orthogonal binding proteins (PYR1, ABI1) that recognize different sites of Mandipropamid, a small molecule. Transfection of neurons with a cDNA that encodes both PYR1-MTD and ABI1-HECT on the same mRNA transcript separated by viral peptide P2A cleavage motifs enables equal stoichiometries of the two halves of iUbiquiNav to be expressed in sensory neurons. In the absence of Mandipropamid, these proteins remain separate (though the PYR1-MTD should still bind to the C-terminus of Nav1.8. In the presence of Mandipropamid, the PYR1-MTD and ABI1-HECT modules should rapidly dimerize, yielding a reconstituted functional UbiquiNav protein that then ubiquitinates Nav1.8. FIG.2K shows the effect of iUbiquiNav on Nav1.8 currents within 1 hour of Mandipropamid45678892.18 ATTORNEY DOCKET NO. YU 8723 PCT application. Following baseline recordings, DRG neurons expressing iUbiquiNav or GFP-control were perfused with 1 µM Mandipropamid. Every 10 seconds, a voltage step to the peak of activation (matched to the particular cell) was applied. Currents are normalized to the peak inward current at sweep 1. Mandipropamid was applied at the 5th sweep. FIGs.3A-3J show that UbiquiNav normalizes nociceptor excitability and delivery and distribution of Nav1.8 channels in hyperexcitable states. FIG.3A shows the effect of UbiquiNav on neuronal hyperexcitability evoked by application of TNF-α. whole-cell current clamp recordings of DRG neurons from 2-4 d old rat pups in response to injection of 200 pA of current for 1 s. Control: transfected with eGFP, incubated with 40 ng / mL DMSO. TNF-α: transfected with eGFP, incubated with 40 ng / mL TNF-α for 24 hours. TNF-α + UbiquiNaV: transfected with UbiquiNaV, incubated with 40 ng / mL TNF-α for 24 hours. Rat pup DRG neurons do not fire readily at baseline. Application of the inflammatory cytokine TNF-α mimics an inflammatory pain state and results in neuronal hyperexcitability. DRG neurons expressing UbiquiNaV do not become hyperexcitable in response to TNF-α exposure. FIG.3B is a bar graph showing AP firing frequency in response to a 1 second current injection of 200 pA. Action potentials fired in response to 200 pA current injection in control (first bar; n=11), TNF- α treated (second bar; n=14) or UbiquiNaV expressing + TNF-α treated (third bar; n=14) DRG neurons. FIG.3C is a bar graph shows that current was injected (for 1 second) in gradually increasing 25 pA steps and the number of overshooting action potentials was counted. Action potential firing threshold in control (first bar; n=11), TNF α treated (2ndbar; n=14) or UbiquiNaV expressing + TNF-α treated (third bar; n=14) DRG neurons. In peripheral sensory neurons, NaV isoforms can be distinguished on the basis of their pharmacologic susceptibility to tetrodotoxin (TTX). Nav isoforms are either sensitive to TTX blockade (TTX-S) or resistant (TTX-R). FIG.3D is a line graph showing repetitive action potential firing in response to gradually increasing current injections (25 pA-500 pA) of 1 s duration in control (first bar; n=11), TNF-α treated (second bar; n=14) or UbiquiNaV expressing + TNF-α treated (third bar; n=14) DRG neurons. FIG.3E is a bar graph showing quantification of somatic Halo-NaV1.8 surface expression in TNF-α incubated neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP-control (first bar; n=27) or UbiquiNaV (second bar; n=19). Halo-NaV1.8 expression at neuronal surfaces in Halo-NaV1.8-expressing neurons incubated with 40 ng / mL TNF-α transfected with eGFP-control or UbiquiNaV. Somatic expressions and axonal expression were measured. FIG.3F is a bar graph showing quantification of axonal Halo-NaV1.8 surface expression in TNF-α incubated neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP- control (first bar; n=17) or UbiquiNaV (second bar; n=20). FIG.3G is a bar graph showing45678892.19 ATTORNEY DOCKET NO. YU 8723 PCT quantification of somatic Halo-NaV1.8 surface expression in TNF-α incubated neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP-control (first bar; n=29) or UbiquiNaV (second bar; n=15). Halo-NaV1.8 expression at neuronal surfaces in Halo-NaV1.8-expressing neurons incubated with 50 nM Paclitaxel transfected with eGFP-control or UbiquiNaV. Somatic expressions and axonal expressions were measured. FIG.3H is a bar graph showing quantification of axonal Halo-NaV1.8 surface expression in TNF-α incubated neurons transfected with Halo-NaV1.8-P2A-mRuby2 and eGFP-control (first bar; n=34) or UbiquiNaV (second bar; n=30). FIG.3I is a bar graph showing quantitation of anterograde vesicular flux of Halo-NaV1.8 channels in neurons incubated with TNF-α and transfected with eGFP-control (first bar; n=16) or UbiquiNaV (second bar; n=15). An axon containing anterogradely trafficking vesicles carrying Halo-NaV1.8 in neurons incubated with TNF-α and transfected with eGFP-control or UbiquiNaV. FIG.3J is a bar graph showing quantitation of anterograde vesicular flux of Halo-NaV1.8 channels in neurons incubated with Paclitaxel and transfected with eGFP-control (first bar; n=16) or UbiquiNaV (second bar; n=15). Bars: Mean ± SEM. ns p> 0.05, * p<0.05, ** p<0.01, *** p<0.001 by the Mann-Whitney U-test (Figures 3B, 3C, 3E, 3F, 3G, 3H, 3I), Student’s unpaired t-test (Figure 3J), or linear mixed- effects modeling with post-hoc comparison (Figure 3D). Multiple comparisons corrected by quantifying the False Discovery Rate and adjusting p-values. FIGs.4A-4P shows that UbiquiNav is selective for Nav1.8. FIG.4A is a schematic of high-throughput screening assay for the unbiased evaluation of UbiquiNaV-mediated effects on human NaV isoforms. Expi293F (suspension) are co-transfected with plasmids encoding eGFP- P2A-NaV channels and either UbiquiNaV-P2A-mCherry or mCherry-control. Fluorescence activated cell sorting is conducted to isolate cells expressing both proteins. Whole cell voltage clamp recordings are achieved using the 384-well Sophion Qube Automated Patch Clamp robot. FIG.4B is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing eGFP-2A-NaV1.1 and either mCherry control (first bar; n=39) or UbiquiNaV-P2A-mCherry (second bar; n=77). FIG.4C is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing eGFP-2A-NaV1.2 and either mCherry control (first bar; n=18) or UbiquiNaV-P2A-mCherry (second bar; n=25). FIG.4D is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing eGFP-2A-NaV1.3 and either mCherry control (first bar; n=34) or UbiquiNaV- P2A-mCherry (second bar; n=45). FIG.4E is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing45678892.110 ATTORNEY DOCKET NO. YU 8723 PCT eGFP-2A-NaV1.4 and either mCherry control (first bar; n=48) or UbiquiNaV-P2A-mCherry (second bar; n=25). FIG.4F is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing eGFP-2A-NaV1.5 and either mCherry control (first bar; n=25) or UbiquiNaV-P2A-mCherry (second bar; n=26). FIG.4G is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS Expi293F cells expressing eGFP-2A-NaV1.6 and either mCherry control (○; n=15) or UbiquiNaV-P2A-mCherry (second bar; n=23). FIG.4H is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post- FACS Expi293F cells expressing eGFP-2A-NaV1.7 and either mCherry control (first bar; n=16) or UbiquiNaV-P2A-mCherry (second bar; n=15). FIG.4I is a bar graph showing the peak inward current densities from automated whole-cell voltage clamp of post-FACS ND7 / 23 cells expressing eGFP-2A-NaV1.8 and either mCherry control (first bar; n=18) or UbiquiNaV-P2A- mCherry (second bar; n=15).1 µM TTX was included in the bath to block endogenous TTX-S channels in ND7 / 23 cells. FIG.4J is a schematic of parallel approaches to elucidate the effects of UbiquiNaV expression on the non-NaV electrogenisome. DRG neurons from NaV1.8-null mice do not fire repetitive action potentials (representative trace, yellow). Two methods restore repetitive firing in these neurons - dynamic clamp insertion of mathematically modeled NaV1.8 conductance (representative blue trace) and transfection of a chimeric NaV1.8 channel (containing the NaV1.7 c-terminus) that is resistant to UbiquiNaV binding (representative purple trace). Addition of UbiquiNaV in each of these systems does not affect AP morphology (representative green traces). FIG.4K is a bar graph showing the resting membrane potential recordings from NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) following dynamic clamp addition of NaV1.8 current. FIG.4L is a bar graph showing the action potential firing thresholds recorded from NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) following dynamic clamp addition of NaV1.8 current. FIG.4M is a line graph of the repetitive action potential firing in response to gradually increasing current injections (25 pA-500 pA) of 1 second duration in NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) following dynamic clamp addition of NaV1.8 current. FIG.4N is a bar graph showing the resting membrane potential recordings from NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) and mCherry-P2A-NaV1.8 / 1.7C. FIG.4O is a bar graph showing the action potential firing thresholds recorded from NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) and mCherry-P2A-NaV1.8 / 1.7C. FIG.4P is a line graph45678892.111 ATTORNEY DOCKET NO. YU 8723 PCT showing the repetitive action potential firing in response to gradually increasing current injections (25 pA-500 pA) of 1 s duration in NaV1.8-null mouse DRG neurons transfected with either eGFP-control (first bar) or UbiquiNaV (second bar) and mCherry-P2A-NaV1.8 / 1.7C. Bars: Mean ± SEM. ns p> 0.05, * p<0.05 by the Mann-Whitney U-test (FIG.4B, 4C, 4D, 4E, 4F, 4I, 4L), Student’s unpaired t-test (FIG.4G, 4H, 4K, 4L, 4N, 4O), or linear mixed-effects modeling with post-hoc comparison (FIG.4M, 4P). Multiple comparisons corrected by quantifying the False Discovery Rate and adjusting p-values. FIGs.5A-5D shows AAV-mediated delivery of UbiquiNaV reduces NaV1.8 currents in vivo. FIG.5A is a schematic showing packaging UbiquiNaV in an AAV9 capsid yields a virus (AAV9-UbiquiNaV) that can infect Human DRG neurons and reduce NaV1.8 current density (first bar; n = 9) vs un-infected control (second bar; n = 9). FIG.5B is a schematic of the automated patch clamp assay for the unbiased electrophysiologic assessment of primary neurons from mice infected with AAV. Following intrathecal injection of a AAV9 (with reporter protein), DRG neurons will become fluorescent as infection proceeds. Using BSA gradients, neruons can be purified and then sorted by FACS – yielding a single cell suspension of neurons all positive AAV9 infection. The suspension can then be assayed by automated patch clamp. FIG.5C is a bar graph showing the peak inward NaV1.8 currents recorded by automated patch clamp of DRG neurons isolated from C57BL / 6 mice 6 weeks post intrathecal injection of AAV9-UbiquiNaV (first bar; n = 13) or AAV9-eGFP (second bar; n = 10). FIG.5D is a bar graph showing the peak inward NaV1.8 current density recorded by automated patch clamp of DRG neurons isolated from C57BL / 6 mice 6 weeks post intrathecal injection of AAV9- UbiquiNaV (first bar; n = 13) or AAV9-eGFP (second bar; n = 10). Bars: Mean ± SEM. ** p<0.01, *** p<0.001 by Mann-Whitney U-test. FIGs.6A-6C show that UbiquiNav requires NaV1.8 to travel in vesicles to the distal axon. FIG.6A is a schematic showing SNAP-UbiquiNav-C942S was generated by linking a SNAPTag enzyme to the HECT domain of UbiquiNav. The inactivation of UbiquiNav was necessary since the assay sought to detect UbiquiNav in anterogradely trafficking vesicles. The active UbiquiNav moiety abrogated transport and accumulation of Nav1.8 channels in the soma and axonal compartments and thus was difficult to evaluate by imaging of distal axons. FIG.6B is a schematic of co-trafficking OPAL experiments. DRG neurons from Nav1.8-null mice were transfected with Halo-Nav1.8 and SNAP-UbiquiNav-C942S and plated in MFCs. JFX650-Halo and JFX554cp-SNAP were applied to the somatic chamber of MFCs. After 25 minutes of labeling, MFCs were washed and taken to a confocal microscope for optical pulse-chase imaging of distal axons. FIG.6C is a bar graph showing co-trafficking of SNAP-UbiquiNav-C942S with45678892.112 ATTORNEY DOCKET NO. YU 8723 PCT Halo-Nav1.8. Axons were analyzed for the presence of vesicles containing SNAP-UbiquiNav- C942S. Vesicles were positive for only SNAP-UbiquiNav-C942S if they did not overlap with fluorescent signal from labeled Halo-Nav1.8 channels. FIGs.7A-7C shows UbiquiNav does not affect Tetrodotoxin-sensitive NaV currents in rat pup DRG neurons. FIG.7A shows a pulse protocol used to isolate TTX-S currents in voltage-clamp of 2-4d old rat pup DRG neurons. While holding at -90 mV, total Na+ current was recorded. Then, the holding potential was changed to -50 to inactivate all TTX-S channels. By reference subtraction of the resulting currents, TTX-S current was quantified. Neurons were transfected with either UbiquiNav or eGFP-control. FIG.7B is a bar graph showing peak inward current density of TTX-S currents from DRG neurons expressing either eGFP control (first bar; n=9) or UbiquiNav (second bar; n=10). n.s.- p>0.05 by Student’s unpaired t-test. FIG.7C shows that current-voltage relationship of TTX-S currents recorded from DRG neurons expressing either eGFP control (n=9) or UbiquiNav (n=10). FIGs.8A-8O showing UbiquiNav is selective for NaV1.8 over other human NaV channels Current-voltage relationships from Expi293 cells expressing human NaV channel isoforms and UbiquiNav. Cells were sorted by Flow Cytometry prior to whole cell voltage- clamp recording on the Sophion Qube 384 well Automated Patch Clamp system. FIG.8A shows comparison of NaV1.1 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8B shows comparison of NaV1.2 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8C shows comparison of NaV1.3 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8D shows comparison of NaV1.4 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8E shows comparison of NaV1.5 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8F shows comparison of NaV1.6 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8G shows comparison of NaV1.7 current density in the presence of UbiquiNav (○) or mCherry control (●). FIG.8H shows that NaV1.8 does not express well in HEK293 cells. ND7 / 23 cells were transfected with NaV1.8 prior to FACS and APC to enable comparison of NaV1.8 current density in the presence of UbiquiNav (○) or mCherry control (●). n.s. p>0.05 by linear mixed-effects modeling. FIG.8I shows dynamic clamp addition of simulated Nav1.8 currents restores repetitive firing properties in DRG neurons from Nav1.8-null mice. DRG neurons from Nav1.8-null mice lose repetitive firing capability. Injection of Nav1.8 currents restores robust repetitive firing behavior. FIG.8J shows the effect of UbiquiNav on the resting membrane potential of DRG neurons from Nav1.8-null mice dynamically clamped with Nav1.8 current. Current clamp with simultaneous dynamic clamp was used to evaluate RMP over a45678892.113 ATTORNEY DOCKET NO. YU 8723 PCT period of 10 seconds in Nav1.8-null DRG neurons expressing UbiquiNav or GFP-control. FIG. 8K shows the effect of UbiquiNav on the threshold of action potential firing of DRG neurons from Nav1.8-null mice dynamically clamped with Nav1.8 current. Current clamp with simultaneous dynamic clamp was used to evaluate the minimum current injection required to initiate an overshooting action potential in Nav1.8-null DRG neurons expressing UbiquiNav or GFP-control. FIG.8L shows that UbiquiNav does not affect the repetitive firing capability of DRG neurons from Nav1.8-null mice dynamically clamped with Nav1.8 current. Current clamp with simultaneous dynamic clamp was used to evaluate the repetitive firing responses of Nav1.8- null DRG neurons expressing UbiquiNav or GFP-control in response to 1 second current injections of 25-500 pA in 25 pA steps. FIG.8M is a schematic showing a Nav1.8 channel with the C-terminus of Nav1.7 yields a Nav1.8 channel that would not be bound by UbiquiNaV. FIG. 8N shows that a Nav1.8 channel with the C-terminus of Nav1.7 exhibiting similar gating properties to WT Nav1.8. Nav1.8 / 1.7C channels support similar TTX-R current densities to WT Nav1.8 channels in ND7 / 23 cells. FIG.8O shows that Nav1.8 / 1.7C is resistant to UbiquiNav- mediated reduction in current density. Replacing the C-terminus of Nav1.8 with that of 1.7 enables the expression of Nav1.8 in HEK293 cells grown in suspension cultures. The previously described FACS to APC assay was used on cells co-transfected with Nav1.8 / 1.7C and either UbiquiNav or mCherry-control. FIGs.9A and 9B shows UbiquiNav has species-dependent affinity for NaV1.8 channels. FIG.9A shows the UbiquiNav warhead is the MTD from rat Sclt1. UbiquiNav can reduce human NaV1.8 current density by around 2-fold as evaluated by automated voltage-clamp in ND7 / 23 cells. *p<0.05 by Student’s t-test. FIG.9B shows replacing the rat Sclt1 MTD with the human SCLT1 MTD yields “human UbiquiNav” (hUbiquiNaV). hUbiquiNav can reduce NaV1.8 current density by around 4-fold as evaluated by automated voltage-clamp in ND7 / 23 cells. ****p<0.05 by Student’s t-test. FIGs.10A-10C shows dynamic clamp addition of NaV1.8 conductance restores repetitive firing in NaV1.8-null mouse DRG neurons. FIG.10A shows dynamic clamp modeling (Hodgkin-Huxley) of NaV1.8 conductance yields simulated NaV1.8 currents (blue trace) that simulate recorded NaV1.8 currents (black trace) with high fidelity. FIG.10B shows NaV1.8-null mouse DRG neurons are incapable of repetitive firing (top trace). Dynamic clamp addition of simulated NaV1.8 conductance restores repetitive firing capability in these neurons (bottom trace). FIG.10C shows quantification of action potentials fired in response to a 300-pA stimulus before (●) and after (●) dynamic clamp addition of Nav1.8 conductance. **** p<0.0001 by Mann-Whitney U-test.45678892.114 ATTORNEY DOCKET NO. YU 8723 PCT FIGs.11A-11C shows that replacing the NaV1.8 C-terminus with the NaV1.7 C- terminus yields a functional NaV1.8 channel resistant to UbiquiNav-mediated degradation. FIG. 11A is a schematic of UbiquiNav binding to the NaV1.8 C-terminus. Swapping the C-terminus of NaV1.8 with the C-terminus of NaV1.7 removes the binding site of UbiquiNaV from the NaV1.8 channel. FIG.11B shows that swapping the C-terminus of NaV1.8 with the C-terminus of NaV1.7 yields a channel that demonstrates a similar current-voltage relationship and expression profile with WT human NaV1.8. FIG.11C shows that NaV1.8 / 1.7C can be expressed in Expi293 cells, unlike WT NaV1.8. NaV1.8 / 1.7C currents are not reduced in the presence of UbiquiNav (●) vs. mCherry control (●). n.s. p>0.05 by linear mixed effects modeling. DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. “Endogenous” refers to any material from or produced inside an organism, cell, tissue or system. “Exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. “Encoding” or “encode” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein, the term “locus” is the specific physical location of a DNA sequence (e.g., of a gene) on a chromosome. It is understood that a locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e., in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of45678892.115 ATTORNEY DOCKET NO. YU 8723 PCT genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples. “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a nonnative environment such as, for example, a host cell. An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes: a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and / or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. A “vector” is a composition of matter which includes an isolated nucleic acid, and which can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of vectors include but are not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” encompasses an autonomously replicating plasmid or a virus. The term is also construed to include non plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, and the like. The term “inhibit” or other forms of the word such as “inhibiting” or “inhibition” means to decrease, hinder or restrain a particular characteristic such as an activity, response, condition, disease, or other biological parameter. It is understood that this is typically in relation to some45678892.116 ATTORNEY DOCKET NO. YU 8723 PCT standard or expected value, i.e., it is relative, but that it is not always necessary for the standard or relative value to be referred to. “Inhibits” can also mean to hinder or restrain the synthesis, expression or function of a protein relative to a standard or control. Inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. “Inhibits” can also include, for example, a 10% reduction in the activity, response, condition, disease, or other biological parameter as compared to the native or control level. Thus, the reduction can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of reduction in between as compared to native or control levels. For example, “inhibits expression” means hindering, interfering with or restraining the expression and / or activity of the gene / gene product pathway relative to a standard or a control. As used herein, the term “polypeptides” includes proteins and functional fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). As used herein, the term “functional fragment” as used herein is a fragment of a full- length protein retaining one or more function properties of the full-length protein. As used herein, the terms “variant” or “active variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties (e.g., functional or biological activity). As used herein, “conservative” amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties. As used herein, “non-conservative” amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered. As used herein, the term “identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptides as determined by the match45678892.117 ATTORNEY DOCKET NO. YU 8723 PCT between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). As used herein, the term “treat” means to prevent, reduce, decrease, or ameliorate one or more symptoms, characteristics or comorbidities of an age-related disease, disorder or condition; to reverse the progression of one or more symptoms, characteristics or comorbidities of an age related disorder; to halt the progression of one or more symptoms, characteristics or comorbidities of an age-related disorder; to prevent the occurrence of one or more symptoms, characteristics or comorbidities of an age-related disorder; to inhibit the rate of development of one or more symptoms, characteristics or comorbidities or combinations thereof. As used herein, the terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, rodents, simians, and humans. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. As used herein, the term “operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will assist the linked protein to be localized at the specific organelle. As used herein, the term “Localization Signal or Sequence or Domain” or “Targeting Signal or Sequence or Domain” are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, intracellular region or cell state. The signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired location. The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to45678892.118 ATTORNEY DOCKET NO. YU 8723 PCT cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention. Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed.45678892.119 ATTORNEY DOCKET NO. YU 8723 PCT Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. II. COMPOSITIONS The disclosed compositions include a fusion protein, herein, UbiquiNav, which is a bifunctional molecule that includes two bioactive domains, a first bioactive domain for selective binding to Nav1.8 channels and a second bioactive domain for catalyzing a ubiquitination reaction to tag the channel for internalization and degradation. In some forms, the first and second bioactive domains are separated by one or more linkers. A. UbiquiNav, R-UbiquiNav and RL-Nav Polypeptides UbiquiNav, R- UbiquiNav or LR-Nav can be represented by the general formula I D1 / D2-L1-D2 / D1, where D1 is a first bioactive domain, D2 is a second bioactive domain, and L1 is an optional linker. iUbiquiNav, i R- UbiquiNav or iLR-Nav is produced from a fusion protein represented by the general formula: D’1-P1-L1-D’2-P2, Where, D’1 is a first bioactive module; D’2 is a second bioactive module,45678892.120 ATTORNEY DOCKET NO. YU 8723 PCT L1 is an optional flexible linker, for example, an amino acid linker, P1 is a first peptide binding domains of the anti-fungal small molecule Mandipropamid, and P2 is a second peptide binding domains of the anti-fungal small molecule Mandipropamid. In iUbiquiNav, the two critical modules of UbiquiNav (MTD – selective binding; HECT of NEDD4L – catalytic) are linked to orthogonal binding proteins (PYR1, ABI1) that recognize different sites of Mandipropamid, a small molecule (FIG.2B). Transfection of neurons with a cDNA that encodes both PYR1-MTD and ABI1-HECT on the same mRNA transcript separated by a peptide cleavage motifs allows equal stoichiometries of the two halves of iUbiquiNav to be expressed in sensory neurons. In the absence of Mandipropamid, these constructs remain separate (though the PYR1-MTD should still bind to the C-terminus of NaV1.8. In the presence of Mandipropamid, the PYR1-MTD and ABI1-HECT modules should rapidly dimerize, yielding a functional UbiquiNav fusion protein that then ubiquitinates NaV1.8. Thus, iUbiquiNav is two separate fusion proteins generally represented by the formulae D’1-P1 and D’2-P2, which are rendered active by coadministration / expression of an agent that dimerizes P1 and P2. In some forms, D1 / D’1 includes an MTD sequence and D2 / D’2 includes the HECT domain is from NEDD4 ubiquitin ligase. In some forms, D1 includes the HECT domain is from NEDD4 ubiquitin ligase and D2 / / D’2 includes an MTD sequence. The MTD sequence and the HECT domain is from NEDD4 ubiquitin ligase are preferably, human. NEDD4 is the founding member of the NEDD4 family of ubiquitin ligases that is evolutionarily conserved in eukaryotes. Ubiquitination plays a crucial role in regulating proteins post-translationally. Ubiquitination involves the covalent attachment of the 8kDa protein ubiquitin to one or more lysine residues in the substrate protein to signal proteins for degradation, altered localization, trafficking or function. Substrate proteins can be mono- ubiquitinated, multi-monoubiquitinated or poly-ubiquitinated, with the type of ubiquitination determining the fate of the protein. Ubiquitin is covalently attached to a protein substrate via an energy dependent three step process, involving an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin protein ligase. The E3 ubiquitin ligase largely determines the substrate specificity of the system and in mammals there are several hundred ubiquitin protein ligases. These can be grouped into two main classes; the RING (Really Interesting New Gene) E3s which mediate the direct transfer of ubiquitin to the substrate, and the HECT (Homologous to E6-AP C-Terminus) E3s which are involved in the transfer of activated ubiquitin from the E2 to the substrate by forming an intermediate complex with the C-terminus of the E3. The disclosed compositions and methods employ ubiquiting ligase catalytic domains. The NEDD4 gene was cloned in 1992 as one of a number of murine Nedd (Neural precursor cell45678892.121 ATTORNEY DOCKET NO. YU 8723 PCT expressed developmentally down-regulated) genes differentially expressed in the central nervous system. At the time of its cloning, the predicted protein had only one known domain – an N- terminal calcium / lipid-binding domain (C2 domain). The presence of three partial repeats of approximately 40 amino acids containing two conserved tryptophan residues in the middle part of the protein was also noted. These repeats, now known to occur in numerous proteins, are widely known as WW domains). Subsequently, the C-terminal region of NEDD4 was found to be similar to human E6-AP, the papilloma virus oncoprotein E6-associated protein. E6-AP was the first discovered ubiquitin-protein ligase, and it was shown to be involved in the E6-mediated ubiquitination of p53. The C-terminus of E6-AP comprising the catalytic domain was named HECT (homologous to the E6-AP C-terminus). E6-AP became the founding member of the HECT type of E3 ubiquitin ligases, of which now there are 29 human members. NEDD4 is a highly evolutionarily conserved protein from yeast to man and was initially cloned as a highly expressed gene in the early embryonic brain. There are 94 orthologues of NEDD4 in the NCBI database, all sharing the same modular structure consisting of an N- terminal C2 domain, 3–4 WW domains and a C-terminal catalytic HECT domain for ubiquitin protein ligation. The HECT domain is a highly conserved domain that includes around 350 amino acids and contains a conserved cysteine residue that forms an intermediate thioester bond with the activated ubiquitin accepted from an E2, before catalyzing the ubiquitination of a lysine in the substrate protein. (Reviewed in Boase, et al., Gene, 557(2):113-122 (2015). i. HECT Catalytic Domain In some forms the HECT catalytic domain is from human NEDD4 ubiquitin ligase, and it is encoded by the following nucleotide sequence: TCCAGAGAATTTAAGCAGAAATATGACTACTTCAGGAAGAAATTAAAGAAACCTGCTGATATCC CCAATAGGTTTGAAATGAAACTTCACAGAAATAACATATTTGAAGAGTCCTATCGGAGAATTAT GTCCGTGAAAAGACCAGATGTCCTAAAAGCTAGACTGTGGATTGAGTTTGAATCAGAGAAAGGT CTTGACTATGGGGGTGTGGCCAGAGAATGGTTCTTCTTACTGTCCAAAGAGATGTTCAACCCCT ACTACGGCCTCTTTGAGTACTCTGCCACGGACAACTACACCCTTCAGATCAACCCTAATTCAGG CCTCTGTAATGAGGATCATTTGTCCTACTTCACTTTTATTGGAAGAGTTGCTGGTCTGGCCGTA TTTCATGGGAAGCTCTTAGATGGTTTCTTCATTAGACCATTTTACAAGATGATGTTGGGAAAGC AGATAACCCTGAATGACATGGAATCTGTGGATAGTGAATATTACAACTCTTTGAAATGGATCCT GGAGAATGACCCTACTGAGCTGGACCTCATGTTCTGCATAGACGAAGAAAACTTTGGACAGACA TATCAAGTGGATTTGAAGCCCAATGGGTCAGAAATAATGGTCACAAATGAAAACAAAAGGGAAT ATATCGACTTAGTCATCCAGTGGAGATTTGTGAACAGGGTCCAGAAGCAGATGAACGCCTTCTT GGAGGGATTCACAGAACTACTTCCTATTGATTTGATTAAAATTTTTGATGAAAATGAGCTGGAG45678892.122 ATTORNEY DOCKET NO. YU 8723 PCT TTGCTCATGTGCGGCCTCGGTGATGTGGATGTGAATGACTGGAGACAGCATTCTATTTACAAGA ACGGCTACTGCCCAAACCACCCCGTCATTCAGTGGTTCTGGAAGGCTGTGCTACTCATGGACGC CGAAAAGCGTATCCGGTTACTGCAGTTTGTCACAGGGACATCGCGAGTACCTATGAATGGATTT GCCGAACTTTATGGTTCCAATGGTCCTCAGCTGTTTACAATAGAGCAATGGGGCAGTCCTGAGA AACTGCCCAGAGCTCACACATGCTTTAATCGCCTTGACTTACCTCCATATGAAACCTTTGAAGA TTTACGAGAGAAACTTCTCATGGCCGTGGAAAATGCTCAAGGATTTGAAGGGGTG (SEQ ID NO:1). Thus, in some forms, the composition includes SEQ ID NO: 1. The peptide encoded by SEQ ID NO: 1 is shown below as SEQ ID NO:2. SREFKQKYDYFRKKLKKPADIPNRFEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKG LDYGGVAREWFFLLSKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAV FHGKLLDGFFIRPFYKMMLGKQITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQT YQVDLKPNGSEIMVTNENKREYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELE LLMCGLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGF AELYGSNGPQLFTIEQWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGV (SEQ ID NO:2). The E3 ubiquitin-protein ligase NEDD4 isoform 1 protein sequence can be found at NCBI Reference Sequence: NP_006145.2, the HECT region of which is shown below as SEQ ID NO:3. In some forms, the HECT sequence includes the amino acids: DFLKARLWIEFDGEKGLDYGGVAREWFFLISKEMFNPYYGLFEYSATDNYTLQINPNSGLCNED HLSYFKFIGRVAGMAVYHGKLLDGFFIRPFYKMMLHKPITLHDMESVDSEYYNSLRWILENDPT ELDLRFIIDEELFGQTHQHELKNGGSEIVVTNKNKKEYIYLVIQWRFVNRIQKQMAAFKEGFFE LIPQDLIKIFDENELELLMCGLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIR LLQFVTGTSRVPMNGFAELYGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEELWDKL QMAIENTQGF (SEQ ID NO:3). Therefore, the compositions typically are, or include SEQ ID NO:2 or SEQ ID NO:3, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2, or a nucleic acid encoding the same. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may45678892.123 ATTORNEY DOCKET NO. YU 8723 PCT be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide. Modifications and changes can be made in the structure of the polypeptides disclosed herein and still have similar characteristics to the original polypeptide (catalyzing a ubiquitination reaction). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Since it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). The polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest. In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Substitution of like amino acids can also be made45678892.124 ATTORNEY DOCKET NO. YU 8723 PCT on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological forms. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (- 2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Forms of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, forms of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.45678892.125 ATTORNEY DOCKET NO. YU 8723 PCT “Identity” and “similarity” can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure. ii. Ubiqutin Ligase Recruiting peptide sequence In some forms, the disclosed fusion proteins include a domain which recruits / binds an endogenous ubiquitin ligase such as E3. E3 ubiquitin ligases are critical in the final stage of the three-step protein ubiquitination process. Initially, E1 ubiquitin-activating enzymes create a thioester bond with ubiquitin, which is then transferred to an E2 ubiquitin-conjugating enzyme. This E2 ubiquitin complex interacts with an E3 ligase that aligns the ubiquitin-loaded E2 and the target protein to facilitate the attachment of ubiquitin to a lysine residue on the substrate. This results in monoubiquitination, which can further undergo successive ubiquitination, forming a Lys48-linked polyubiquitin chain. When this chain reaches a length of four ubiquitins, it signals the protein for degradation by the 26S proteasome. The human genome contains two E1 enzymes, 38 E2s, and over 600 E3 ligases. This process is reviewed in further detail in Baird and Yamamato, Molecular and Cellular Biology, 40(13): e00099-20 (2020). In one exemplary form, the compositions include a recruiting sequence for the ligase Kelch-like ECH-associated protein 1 (KEAP1) or a functional fragment or variant thereof. KEAP1 is a component of an E3 ubiquitin ligase. In the KEAP1- CUL3-RBX1 E3 ubiquitin ligase complex, KEAP1 functions as the substrate adaptor, RBX1 binds to the ubiquitin loaded E2-ubiquitin conjugating enzyme, and CUL3 (Cullin 3) provides the scaffold which joins KEAP1 and RBX1 (Ring Box Protein 1). Together, the complex functions to correctly orientate the NRF2-bound KEAP1 and the E2-bound RBX1 to facilitate ubiquitination of NRF2. A more detailed review of the structure and function of KEAP1 can be found in Baird and Yamamato, Molecular and Cellular Biology, 40(13): e00099-20 (2020). Human KEAP1 protein (Uniprot ID: Q14145·KEAP1_Human) has the amino acid sequence represented by SEQ ID NO:25. MQPDPRPSGAGACCRFLPLQSQCPEGAGDAVMYASTECKAEVTPSQHGNRTFSYTLEDHTKQAF GIMNELRLSQQLCDVTLQVKYQDAPAAQFMAHKVVLASSSPVFKAMFTNGLREQGMEVVSIEGI HPKVMERLIEFAYTASISMGEKCVLHVMNGAVMYQIDSVVRACSDFLVQQLDPSNAIGIANFAE45678892.126 ATTORNEY DOCKET NO. YU 8723 PCT QIGCVELHQRAREYIYMHFGEVAKQEEFFNLSHCQLVTLISRDDLNVRCESEVFHACINWVKYD CEQRRFYVQALLRAVRCHSLTPNFLQMQLQKCEILQSDSRCKDYLVKIFEELTLHKPTQVMPCR APKVGRLIYTAGGYFRQSLSYLEAYNPSDGTWLRLADLQVPRSGLAGCVVGGLLYAVGGRNNSP DGNTDSSALDCYNPMTNQWSPCAPMSVPRNRIGVGVIDGHIYAVGGSHGCIHHNSVERYEPERD EWHLVAPMLTRRIGVGVAVLNRLLYAVGGFDGTNRLNSAECYYPERNEWRMITAMNTIRSGAGV CVLHNCIYAAGGYDGQDQLNSVERYDVETETWTFVAPMKHRRSALGITVHQGRIYVLGGYDGHT FLDSVECYDPDTDTWSEVTRMTSGRSGVGVAVTMEPCRKQIDQQNCTC (SEQ ID NO:25). In some forms, the compositions include a recruiting sequence for the KEAP1 ligase, or a functional fragment or variant thereof. A recruiting peptide sequence as used herein refers to refers to a specific segment within a protein that facilitates the targeted / specific interaction between the protein containing the recruiting sequence and another protein e.g., KEAP1 ligase. An exemplary recruiting sequence for KEAP1 is LDPETGEYL (SEQ ID NO:26). Thus, in some forms, the compositions include SEQ ID NO:26, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:26, or a nucleic acid encoding the same. In some forms, the composition incorporates a degron. E3s bind their substrates directly via E3 binding sites present on the surface of substrates. These binding sites are called degrons. Degrons are short linear motifs, bound by E3 ubiquitin ligase to target protein substrates to be degraded by the ubiquitin-proteasome system. The interaction between E3 and degron determines the specificity of the degradation process. Degrons are preferentially located in disordered regions and are molecular recognition features (MoRFs) that undergo disorder-to- order transition upon binding to E3s. Degrons are typically regulated by post-translational modifications (PTMs), which control the interaction with E3s in response to environmental and cellular cues. Degrons mediate the ubiquitination of substrates, and the resulting Ub-sites are usually located within 20 amino acids (AAs) distant from the degron. A fundamental property of degrons is their transferability: in most cases, transplantation of a degron to a protein accelerates the degradation of a protein. Degrons are reviewed in further detail in Hou, et al., BMC Biology, 20, Article Number 162 (2022). An exemplary degron is RRRG (SEQ ID NO:27). Thus, in some forms, the compositions include SEQ ID NO:27, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:27, or a nucleic acid encoding the same. Other targeting peptides include MLAP(OH)YIPM (SEQ ID NO:36); LAP(OH)YI (SEQ ID NO:37), ALAPYIP (SEQ ID NO:38); DRHDS(P)GLDS(P)M (SEQ ID NO:39) and cyclic RGDyK (SEQ ID NO:40) (Wang, et al., Biomater Res 27, 72 (2023))45678892.127 ATTORNEY DOCKET NO. YU 8723 PCT iii. Lysosome Recruiting Domains for NaV1.8 The above proposed recruitment domains leverage the same ubiquitin / proteasome axis as UbiquiNav. However, in other forms, rather than a domain for a ubiquitin ligase, the composition incorporates a lysosome recruiting sequence which brings Nav1.8 directly to the lysosome by leveraging the action of one or more non-ubiquitin proteins. Therefore, in some forms, the compositions include one or more recruitment domains which hijack the endogenous degradative machinery of the cells for the targeted degradation of Nav1.8. More specifically, the one or more lysosome recruitment domains are ubiquitin-independent and take advantage of the lysosome / chaperone mediated autophagy pathways. An exemplary lysosome recruitment domain brings Nav1.8 to the lysosome directly by leveraging the action of the protein LAMP2A (lysosomal associated membrane protein 2). An exemplary lysosomal targeting sequence isKFERQKILDQRFFE (SEQ ID NO:28). Thus, in some forms, the compositions include SEQ ID NO:28, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:28, or a nucleic acid encoding the same. Another lysosome recruitment domain brings Nav1.8 directly to the lysosome by leveraging the action of the proteins hsc70 (heat shock protein 70) and LAMP2A. exemplary lysosomal targeting / recruitment peptide sequence is MDFSGLSLIKLKKQ (SEQ ID NO:29). Thus, in some forms, the compositions include SEQ ID NO:29, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29, or a nucleic acid encoding the same. In some forms, the lysosomal recruiting sequence is KFERQ (SEQ ID NO:35) or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29, or a nucleic acid encoding the same. iv. MTD Domain The bioactive domain for selective binding to Nav1.8 channels is the preferably MTD domain of clathrin linker-1 (SCLT-1, previously called clathrin-associated protein 1A, CAP-1A). SCLT1 acts as a linker protein between the voltage-gated sodium channel Na(v)1.8 (SCN10A; 604427) and clathrin (Liu, et al. Molec. Cell. Neurosci.28: 636-649, 2005). Using yeast 2-hybrid analysis with the C terminus of rat Scn10a as bait to screen a rat brain cDNA library, Liu et al. (2005) cloned rat Sclt1, which they called CAP1A. By database analysis, they identified the mouse and human SCLT1 sequences. The deduced 688-amino acid human SCLT1 protein (GenBank accession number BAB70876) shares 76% sequence identity with the rat45678892.128 ATTORNEY DOCKET NO. YU 8723 PCT protein. SCLT1 contains a myosin tail domain (MTD), a vacuolar ATPase homology domain (V- pump), and dileucine motifs in the C terminal terminus. SCLT1 lacks a signal peptide and transmembrane segments. In some forms, the MTD domain is from a human SCLT-1 protein. The deduced amino acid sequence of human CAP-1A is shown below (Liu, et al. Molec. Cell. Neurosci.28: 636-649, 2005, FIG.1). Human SCLT1 protein sequence (NP_ 653244.2) is shown below. The Myosin tail domain (MTD) is shown in italics, bold font (and broken underlined) MAAEIDFLREQNRRLNEDFRRYQMESFSKYSSVQKAVCQGEGDDTFENLVFDQSFLAPLVTEYD KKKEKDVVSAHGREEASDRRLQQLQSSIKQLEIRLCVTIQEANQLRTENTHLEKQTRELQAKCN ELENERYEAIVRARNSMQLLEEANLQKSQALLEEKQKEEDIEKMKETVSRFVQDATIRTKKEVA NTKKQCNIQISRLTEELSALQMECAEKQGQIERVIKEKKAVEEELEKIYREGRGNESDYRKLEE MHQRFLVSERSKDDLQLRLTRAENRIKQLETDSSEEISRYQEMIQKLQNVLESERENCGLVSEQ RLKLQQENKQLRKETESLRKIALEAQKKAKVKISTMEHEFSIKERGFEVQLREMEDSNRNSIVE LRHLLATQQKAANRWKEETKKLTESAEIRINNLKSELSRQKLHTQELLSQLEMANEKVAENEKL ILEHQEKANRLQRRLSQAEERAASASQQLSVITVQRRKAASLMNLENI (SEQ ID NO:4). Thus, the compositions typically are, or include SEQ ID NO:5. or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5, or a nucleic acid encoding the same, where SEQ ID NO:5 is VTEDLQGQMKKKEKDVVSAHGREEASDRRLQQLQSSIKQLEIRLCVTIQEANQLRTENTHLEKQ TRELQAKCNELENERYEAIVRARNSMQLLEEANLQKSQALLEEKQKEEDIEKMKETVSRFVQDA TIRTKKEVANTKKQCNIQISRLTEELSALQMECAEKQGQIERVIKEKKAVEEELEKI (SEQ ID NO:5). The human SCLT1 cDNA nucleotide sequence (NM_144643.4) is shown below and the sequence encoding MTD is italic and bold font (double underlined). ATGGCTGCAGAAATCGACTTTCTGAGAGAGCAAAATCGAAGACTAAATGAAGATTTTAGGCGGT ATCAAATGGAAAGTTTTTCCAAATATTCATCTGTACAGAAAGCTGTCTGCCAAGGAGAAGGAGA CGACACATTTGAAAACCTAGTATTTGACCAAAGCTTTTTAGCTCCTCTTGTTACTGAGTATGAT AAACACCTAGGAGAACTAAATGGGCAGCTGAAATATTACCAGAAACAGGTGGGTGAGATGAAAT TACAACTTGAAAATGTCATCAAGGAAAATGAAAGGTTGCACAGTGAATTAAAAGATGCTGTTGA AAAAAAATTGGAGGCCTTTCCCCTGGGCACAGAGGTAGGAACTGACATATATGCAGATGATGAA 45678892.129 ATTORNEY DOCKET NO. YU 8723 PCT ACAGTCAGAAACCTTCAAGAACAATTGCAGCTAGCCAATCAAGAAAAAACTCAGGCTGTGGAAC TCTGGCAGACTGTTTCTCAGGAGTTGGACAGACTACACAAGCTTTACCAGGAACATATGACTGA GGCCCAGATTCATGTATTTGAAAGTCAAAAACAAAAGGATCAGCTATTTGATTTTCAACAACTG ACCAAACAACTTCATGTTACTAATGAGAACATGGAAGTGACTAACCAACAGTTTCTGAAAACAG TAACTGAACAAAGTGTGATAATCGAACAACTCCGAAAAAAACTTAGGCAAGCCAAATTAGAGCT GAGAGTTGCTGTAGCAAAAGTGGAAGAGTTAACTAATGTGACTGAAGATCTGCAGGGACAGATG AAAAAGAAGGAGAAGGATGTGGTGTCTGCCCATGGAAGAGAGGAAGCATCAGATAGGCGTTTAC AGCAGTTACAGTCTAGTATAAAACAATTAGAAATAAGATTATGTGTGACAATCCAAGAAGCCAA CCAATTAAGAACCGAAAATACACATCTGGAAAAACAGACCAGAGAGCTACAAGCAAAGTGCAAT GAATTAGAAAATGAGAGATATGAGGCTATTGTAAGAGCCAGAAATAGCATGCAACTCTTAGAAG AAGCTAACCTTCAAAAAAGTCAGGCTCTACTTGAGGAGAAGCAAAAAGAAGAAGACATAGAGAA AATGAAAGAGACAGTTTCTCGGTTTGTACAAGATGCTACCATAAGAACCAAGAAAGAAGTTGCA AACACCAAAAAACAATGTAATATACAAATTTCTCGATTAACAGAAGAACTTTCAGCCCTTCAAA TGGAGTGTGCTGAAAAACAAGGCCAAATTGAACGAGTCATTAAGGAAAAAAAAGCAGTGGAAGA AGAACTAGAAAAGATTTACCGTGAAGGCAGAGGAAATGAGAGTGATTACAGAAAACTGGAAGAA ATGCACCAAAGATTCCTGGTTTCAGAGCGTTCAAAAGATGATCTTCAGCTAAGACTTACGAGAG CAGAAAATAGAATAAAACAACTTGAAACTGACTCCTCAGAAGAAATATCACGTTACCAAGAAAT GATTCAGAAACTTCAAAATGTATTGGAGTCTGAGAGAGAGAACTGTGGGCTTGTCAGTGAACAA AGGCTAAAACTTCAGCAAGAAAATAAACAGTTACGGAAAGAGACTGAGAGTTTAAGGAAGATTG CCCTGGAGGCTCAAAAAAAAGCCAAAGTAAAGATCAGTACAATGGAACATGAATTTTCAATAAA GGAACGTGGATTTGAAGTTCAATTGAGAGAGATGGAAGACAGTAATAGAAATTCCATTGTTGAA CTGAGGCATCTCCTAGCGACTCAACAGAAGGCAGCCAATAGGTGGAAAGAAGAAACGAAGAAAC TTACTGAAAGTGCAGAAATTAGAATCAATAATCTAAAGAGTGAGCTGAGTCGACAGAAACTTCA TACCCAAGAGCTGCTTTCTCAGCTGGAAATGGCAAATGAAAAGGTAGCTGAGAATGAAAAGCTA ATTCTAGAGCATCAAGAAAAAGCCAACAGACTTCAAAGGCGTCTAAGTCAGGCAGAAGAGAGAG CTGCTTCAGCTTCCCAGCAGCTCAGTGTGATTACAGTGCAGAGAAGAAAAGCAGCCTCCCTGAT GAATCTGGAAAATATT (SEQ ID NO:22). v. Linkers The disclosed pain active agents includes one or more optional flexible linker sequence between the first and second bioactive module. The flexible peptide linker sequence is at least 2 amino acids in length. A “flexible linker” herein refers to a peptide or polypeptide containing two or more amino acid residues joined by peptide bonds that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker. Exemplary flexible peptides / polypeptides include, but are not45678892.130 ATTORNEY DOCKET NO. YU 8723 PCT limited to, the amino acid sequencesGSTG (SEQ ID NO: 6); GSGSGS (SEQ ID NO:7),ASGGGS (SEQ ID NO:8),GGGGSGGGGSGGGGS (SEQ ID NO:9),GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:10), SGLRSAT (SEQ ID NO:32), and GSGSGSGS (SEQ ID NO:11). Additional peptides include GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:12), which can be included in the construct for iUbiquiNav, iR-UbiquiNav or iLR-Nav. Additional flexible peptide / polypeptide sequences are well known in the art. In one embodiment, L1 is GSTG (SEQ ID NO:6). Additional sequences include: hNedd4L nucleotide sequence: NM_001144967; hNedd4L protein sequence: NP_001138439.1 for Nedd4L [isoform 1]; rat Sclt1 nucleotide sequence: NM_153740 and rat Sclt1 protein sequence: NP_714962.1. vi. Heterologous Sequences Heterologous functional elements that can be associated with, linked, conjugated, or otherwise attached directly or indirectly to the disclosed fusion polypeptide sequence, a nucleic acid, and / or to a nanoparticle or other delivery vehicle. Such molecules include, but are not limited to, protein transduction domains, fusogenic peptides, targeting molecules, and sequences that enhance protein expression and / or isolation. a. Protein Transduction Domains In some embodiments, the disclosed pain active agents, a nucleic acid, and / or to a nanoparticle or other delivery vehicle includes a protein transduction domain (PTD). As used herein, a “protein transduction domain” or PTD refers to a polypeptide, polynucleotide, carbohydrate, organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In preferred embodiments, the protein transduction domain is a polypeptide. A protein transduction domain can be a polypeptide including positively charged amino acids. Thus, some embodiments include PTDs that are cationic or amphipathic. Protein transduction domains (PTD), also known as a cell penetrating peptides (CPP), are typically polypeptides including positively charged amino acids. PTA can be short basic peptide sequences such as those present in many cellular and viral proteins. PTDs are known in the art, and include but are not limited to small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P., Trends in Biotechnology (11):498-503 (2003)). Although several PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, Cell, 55(6):1189-93(1988)) protein of HIV and Antennapedia transcription factor from Drosophila,45678892.131 ATTORNEY DOCKET NO. YU 8723 PCT whose PTD is known as Penetratin (Derossi et al., J Biol Chem., 269(14):10444-50 (1994)). Exemplary protein transduction domains include polypeptides with 11 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. Penetratin and other derivatives of peptides derived from antennapedia (Cheng, et al., Biomaterials, 32(26):6194-203 (2011) can also be used. Penetratin in which additional Args are added, further enhances uptake and endosomal escape, and IKK NBD, which has an antennapedia domain for permeation as well as a domain that blocks activation of NFkB and has been used safely in the lung for other purposes (von Bismarck, et al., Pulmonary Pharmacology & Therapeutics, 25(3):228-35 (2012), Kamei, et al., Journal Of Pharmaceutical Sciences, 102(11):3998-4008 (2013)). The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices. Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia. TAT protein consists of 86 amino acids and is involved in the replication of HIV-1. The TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ ID NO:20)) of the parent protein that appears to be critical for uptake. Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO:21) has been shown to be a PTD. In the current literature TAT has been favored for fusion to proteins of interest for cellular import. Several modifications to TAT, including substitutions of Glutamine to Alanine, i.e., Q ^ A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al., Proc Natl Acad Sci U S A., 97(24):13003-8 (2000)) to up to 33-fold in mammalian cells. (Ho et al., Cancer Res., 61(2):474-7 (2001)). The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 11-arginine stretch was several orders of magnitude more efficient as an intercellular delivery vehicle. Therefore, PTDs can include a sequence of multiple arginine residues, referred to herein as poly-arginine or poly-ARG. In some embodiments the sequence of arginine residues is consecutive. In some embodiments the sequence of arginine residues is non-consecutive. A poly-ARG can include at least 7 arginine residues, more preferably at least 8 arginine residues, most preferably at least 11 arginine residues. In some embodiments, the poly-ARG includes between 7 and 15 arginine residues, preferably between 8 and 15 arginine residues, or any specific integer therebetween. In some embodiments the poly- ARG includes between 7 and 15, more preferably between 8 and 15 consecutive arginine residues. An example of a poly-ARG is RRRRRRR (SEQ ID NO:13). Additional exemplary PTDs include but are not limited to, RQIKIWFQNRRMKWKK (SEQ ID NO:33), mTAT (HIV-1 (with histidine modification) HHHHRKKRRQRRRRHHHHH (SEQ ID NO:14) (Yamano, et al., J45678892.132 ATTORNEY DOCKET NO. YU 8723 PCT Control Release, 152:278–285 (2011)); bPrPp (Bovine prion) MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (SEQ ID NO:15) (Magzoub, et al., Biochem Biophys Res Commun., 348:379–385 (2006)); and MPG (Synthetic chimera: SV40 Lg T. Ant.+HIV gb41 coat)GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:16) (Endoh, et al., Adv Drug Deliv Rev., 61:704–709 (2009)). Short, non-peptide polymers that are rich in amines or guanidinium groups are also capable of carrying molecules across biological membranes. A “fusogenic peptide” is any peptide with membrane destabilizing abilities. In general, fusogenic peptides have the propensity to form an amphiphilic alpha-helical structure when in the presence of a hydrophobic surface such as a membrane. The presence of a fusogenic peptide induces formation of pores in the cell membrane by disruption of the ordered packing of the membrane phospholipids. Some fusogenic peptides act to promote lipid disorder and, in this way, enhance the chance of merging or fusing of proximally positioned membranes of two membrane enveloped particles of various nature (e.g. cells, enveloped viruses, liposomes). Other fusogenic peptides may simultaneously attach to two membranes, causing merging of the membranes and promoting their fusion into one. Examples of fusogenic peptides include a fusion peptide from a viral envelope protein ectodomain, a membrane-destabilizing peptide of a viral envelope protein membrane-proximal domain from the cytoplasmic tails. Other fusogenic peptides often also contain an amphiphilic region. Examples of amphiphilic-region containing peptides include: melittin, magainin, the cytoplasmic tail of HIV1 gp41, microbial and reptilian cytotoxic peptides such as bomolitin 1, pardaxin, mastoparan, carboline, cecropin, entamoeba, and staphylococcal alpha-toxin; viral fusion peptides from (1) regions at the N terminus of the transmembrane (TM) domains of viral envelope proteins, e.g. HIV-1, SIV, influenza, polio, rhinovirus, and coxsackie virus; (2) regions internal to the TM ectodomain, e.g. semliki forest virus, sindbis virus, rota virus, rubella virus and the fusion peptide from sperm protein PH-30: (3) regions membrane-proximal to the cytoplasmic side of viral envelope proteins e.g. in viruses of avian leukosis (ALV), Feline immunodeficiency (FIV), Rous Sarcoma (RSV), Moloney murine leukemia virus (MoMuLV), and spleen necrosis (SNV). Without being bound by theory, it is believed that following an initial ionic cell-surface interaction, some polypeptides containing a protein transduction domain are rapidly internalized by cells via lipid raft–dependent micropinocytosis. For example, transduction of a TAT-fusion protein was found to be independent of interleukin-2 receptor / raft-, caveolar- and clathrin- mediated endocytosis and phagocytosis (Wadia, et al., Nature Medicine, 10:310-315 (2004), and Barka, et al., J. Histochem. Cytochem., 48(11):1453-60 (2000). Therefore, in some embodiments45678892.133 ATTORNEY DOCKET NO. YU 8723 PCT the polypeptides include an endosomal escape sequence that enhances escape of the polypeptide from macropinosomes. In some embodiments the endosomal escape sequence is part of, or consecutive with, the protein transduction domain. In some embodiments, the endosomal escape sequence is non-consecutive with the protein transduction domain. In some embodiments the endosomal escape sequence includes a portion of the hemagglutinin peptide from influenza (HA). The efficiency of nanoparticle delivery systems can also be improved by the attachment of functional ligands to the NP surface. Potential ligands include, but are not limited to, small molecules, cell-penetrating peptides (CPPs), targeting peptides, antibodies or aptamers (Yu, et al., PLoS One., 6:e24077 (2011), Cu, et al., J Control Release, 156:258–264 (2011), Nie, et al., J Control Release, 138:64–70 (2009), Cruz, et al., J Control Release, 144:118–126 (2010)). Attachment of these moieties serves a variety of different functions, such as inducing intracellular uptake, endosome disruption, and delivery of the plasmid payload to the nucleus. There have been numerous methods employed to tether ligands to the particle surface. One approach is direct covalent attachment to the functional groups on PLGA NPs (Bertram, Acta Biomater.5:2860–2871 (2009). Another approach utilizes amphiphilic conjugates like avidin palmitate to secure biotinylated ligands to the NP surface (Fahmy, et al., Biomaterials, 26:5727– 5736 (2005), Cu, et al., Nanomedicine, 6:334–343 (2010)). This approach produces particles with enhanced uptake into cells, but reduced pDNA release and gene transfection, which is likely due to the surface modification occluding pDNA release. In a similar approach, lipid-conjugated polyethylene glycol (PEG) is used as a multivalent linker of penetratin, a CPP, or folate (Cheng, et al., Biomaterials, 32:6194–6203 (2011)). b. Targeting Signal or Domain In some embodiments the disclosed fusion polypeptide, a nucleic acid encoding the disclosed fusion polypeptide and / or to a nanoparticle or other delivery vehicle containing the fusion polypeptide / nucleic acid encoding molecule is modified to include one or more targeting signals or domains. The targeting signal can include a sequence of monomers that facilitates in vivo localization of the molecule. The compositions disclosed herein can be modified to target a specific cell type or population of cells. In one embodiment, the targeting signal binds to its ligand or receptor which is located on the surface of a target cell such as to bring the composition and cell membranes sufficiently close to each other to allow penetration of the composition into the cell.45678892.134 ATTORNEY DOCKET NO. YU 8723 PCT In some embodiments, the targeting molecule is an antibody or antigen binding fragment thereof, an antibody domain, an antigen, a cell receptor, a cell surface receptor, a cell surface adhesion molecule, a viral envelope protein and a peptide selected by phage display that binds specifically to a defined cell. Targeting a polypeptide of interest to specific cells can be accomplished by modifying the polypeptide of interest to express specific cell and tissue targeting signals. These sequences target specific cells and tissues. In some embodiments the interaction of the targeting signal with the cell does not occur through a traditional receptor: ligand interaction. The eukaryotic cell comprises a number of distinct cell surface molecules. The structure and function of each molecule can be specific to the origin, expression, character and structure of the cell. Determining the unique cell surface complement of molecules of a specific cell type can be determined using techniques well known in the art. One skilled in the art will appreciate that the tropism of the proteins of interest described can be altered by changing the targeting signal. In one specific embodiment, compositions are provided that enable the addition of cell surface antigen specific antibodies to the composition for targeting the delivery of polynucleotide-binding polypeptide. It is known in the art that nearly every cell type in a tissue in a mammalian organism possesses some unique cell surface receptor or antigen. Thus, it is possible to incorporate nearly any ligand for the cell surface receptor or antigen as a targeting signal. For example, peptidyl hormones can be used a targeting moiety to target delivery to those cells which possess receptors for such hormones. Chemokines and cytokines can similarly be employed as targeting signals to target delivery of the complex to their target cells. A variety of technologies have been developed to identify genes that are preferentially expressed in certain cells or cell states and one of skill in the art can employ such technology to identify targeting signals which are preferentially or uniquely expressed on the target tissue of interest. c. Additional Sequences The compositions can optionally include additional sequences or moieties, including, but not limited to linkers and purification tags. In a preferred embodiment the purification tag is a polypeptide. Polypeptide purification tags are known in the art and include, but are not limited to His tags which typically include six or more, typically consecutive, histidine residues; FLAG tags, which typically include the sequence DYKDDDDK (SEQ ID NO:17); haemagglutinin (HA) for example, YPYDVP (SEQ ID NO:34); MYC tag for example ILKKATAYIL (SEQ ID NO:18) or EQKLISEEDL (SEQ ID NO:19). Methods of using purification tags to facilitate protein purification are known in the art.45678892.135 ATTORNEY DOCKET NO. YU 8723 PCT Purifications tags can be N-terminal or C-terminal to a protein. The purification tags N- terminal to the recombinant protein can be separated from the polypeptide of interest at the time of the cleavage in vivo. Therefore, purification tags N-terminal to the recombinant protein can be used to remove the recombinant protein from a cellular lysate following expression and extraction of the expression or solubility enhancing amino acid sequence but cannot be used to remove the polypeptide of interest. Purification tags C-terminal to the recombinant protein can be used to remove the polypeptide of interest from a cellular lysate following expression of the recombinant protein but cannot be used to remove the expression or solubility enhancing amino acid sequence. Purification tags that are C-terminal to the expression or solubility enhancing amino acid sequence can be N-terminal to, C-terminal to, or incorporated within the sequence of the polypeptide of interest. Molecular biology techniques have developed so that therapeutic proteins can be genetically engineered to be expressed by microorganisms. The gram-negative bacterium, Escherichia coli, is a versatile and valuable organism for the expression of therapeutic proteins. Although many proteins with therapeutic or commercial uses can be produced by recombinant organisms, the yield and quality of the expressed protein are variable due to many factors. For example, heterologous protein expression by genetically engineered organisms can be affected by the size and source of the protein to be expressed, the presence of an affinity tag linked to the protein to be expressed, codon biasing, the strain of the microorganism, the culture conditions of microorganism, and the in vivo degradation of the expressed protein. Some of these problems can be mitigated by fusing the protein of interest to an expression or solubility enhancing amino acid sequence. Exemplary expression or solubility enhancing amino acid sequences include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and a small ubiquitin-related modifier (SUMO). In some embodiments, the compositions disclosed herein include expression or solubility enhancing amino acid sequence. In some embodiments, the expression or solubility enhancing amino acid sequence is cleaved prior administration of the composition to a subject in need thereof. The expression or solubility enhancing amino acid sequence can be cleaved in the recombinant expression system, or after the expressed protein in purified. B. Nucleic acids 1. Isolated Nucleic Acid Molecules Isolated nucleic acid sequences encoding the disclosed fusion polypeptides are disclosed. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic45678892.136 ATTORNEY DOCKET NO. YU 8723 PCT acids that normally flank one or both sides of the nucleic acid in a mammalian genome. An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In some forms the Herpes virus is, a replication-defective HSV-1 vector derived from wild-type HSV-1 through the deletion of two copies of the viral immediate-early gene ICP4, rendering the vector replication incompetent; in addition, ICP22 was also deleted to reduce the cytotoxic effect (Gurevich, et al., Nature Medicine 28:780–788 (2022)). In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. Nucleic acids can be in sense or antisense orientation or can be complementary to a reference sequence encoding a disclosed fusion polypeptide. Thus, nucleic acids encoding SEQ ID NOs:2, and 5, and fragments and variants thereof, in sense and antisense, and in single stranded and double stranded forms, are provided. Also provided are nucleic acid encoding therapeutic polypeptides for the treatment of other diseases and disorders characterized by high levels of mTOR signaling, in sense and antisense, and in single stranded and double stranded forms. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of nucleic acid. Modifications at the base moiety can include deoxy uridine for deoxythymidine, and 5-methyl-2’-deoxycytidine or 5- bromo-2’-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxy phosphate backbone is replaced by a pseudopeptide backbone, and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid45678892.137 ATTORNEY DOCKET NO. YU 8723 PCT Drug Dev.7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem.4:5-23. In addition, the deoxy phosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. 2. Vectors The application also relates to vectors including an isolated polynucleotide encoding a fusion polypeptide and / or therapeutic polypeptides for the treatment of pain, including chronic pain. As used herein, a “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and / or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). A vector can be a DNA vector or an RNA vector. In some embodiments, a vector is a DNA plasmid. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure. A vector of the application can be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Vectors can contain a variety of regulatory sequences. As used herein, the term “regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and / or transport of the nucleic acid or one of its derivatives (i.e. mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability). In some embodiments, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g., a linear covalently closed DNA, e.g., a linear covalently45678892.138 ATTORNEY DOCKET NO. YU 8723 PCT closed double stranded DNA molecule. Preferably, a non-viral vector is a DNA plasmid. A “DNA plasmid”, which is used interchangeably with “DNA plasmid vector,” “plasmid DNA” or “plasmid DNA vector,” refers to a double-stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for production and / or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and / or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC®. complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and / or expression in insect cells; pcDNA™. or pcDNA3™ (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and / or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and / or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)). Preferably, a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNA™, pcDNA3™, pVAX, pVAX-1, ADVAX, NTC8454, etc. In some embodiments, an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is a commonly used plasmid in DNA vaccines and contains a strong human immediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA). pVAX- 1 further contains a pUC origin of replication and a kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.45678892.139 ATTORNEY DOCKET NO. YU 8723 PCT The vector can also be a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which include an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding a fusion polypeptide, an active fragment or variant thereof as described herein), arenavirus such as lymphocytic choriomeningitis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain, etc. In some embodiments, the viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. A recombinant viral vector can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. A polynucleotide encoding a fusion polypeptide of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure. In some forms, the viral vector is selected from the group consisting of a lentiviral vector, an Adeno-associated virus (AAV) vector, an adenovirus vector, a Herpes Simplex virus (HSV) vector, a vesicular stomatitis (VSV) vector, a human Bocavirus vector (hBoV), and a chimeric vector comprising a combination of any two or more of an Adeno-associated virus (AAV)45678892.140 ATTORNEY DOCKET NO. YU 8723 PCT vector, a Herpes Simplex virus (HSV) vector, a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV). In some forms, the vector is an adenoviral vector. Suitable adenoviral vectors include but are not limited to Ad2, Ad3, Ad4, Ad5, Ad7, Ad11, Ad35, and Ad48. In some forms, the vector is an AAV vector. Adeno-associated viruses (AAVs) are widely used vectors for gene therapy due to their ability to deliver genetic material to a variety of cell types with minimal immunogenicity. In some forms, ~1015intact AAV particles carrying DNA cargo of therapeutic gene of interest are required for a single dose. These particles are produced using human or insect cell lines along with defective particles. In some forms, the AAV vector is any AAV vector that can be used to target sensory neurons e.g. central nervous system neurons and peripheral sensory neurons. Exemplary AAV vectors include but are not limited to AAV1, AAV4, AAV5, AAV6, AAV8, AAV9 and derived- AAV9 containing viruses. Other AAV vectors include AAVrh10, AAVhu37, and AAVhu68. For example, Shin, et al., Journal of Clinical Investigation, 134(13), (2024), delivered a DRG injection of AAV6-encoded NaViPA1 (Nav1.7) for the treatment of neuropathic pain induced by tibial nerve injury (TNI). The vectors, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), can include any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the fusion polypeptide encoded by the polynucleotide sequence of the vector. 3. Regulatory Elements Any of the disclosed nucleic acids, including RNAs and DNAs such as DNA vectors can include one or more regulatory elements. Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc. An isolated include acid can be, and a vector can include one or more expression cassettes. An “expression cassette” is part of a nucleic acid such as a vector that directs the cellular machinery to make RNA and protein. An expression cassette typically includes three components: a promoter sequence, an open reading frame, and a 3'-untranslated region (UTR) optionally including a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains a coding sequence of a protein of interest (e.g., the disclosed fusion polypeptides, other therapeutic polypeptides, etc.) from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding the disclosed fusion polypeptides or other therapeutic polypeptides.45678892.141 ATTORNEY DOCKET NO. YU 8723 PCT As used herein, the term “operably linked” is to be taken in its broadest reasonable context and refers to a linkage of polynucleotide (or polypeptide, etc.) elements in a functional relationship. A polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application. a. Promoters The disclosed nucleic acids, including vectors, can include a promoter sequence, preferably within an expression cassette, to control expression of a fusion polypeptide or other therapeutic polypeptide of interest. The term “promoter” is used in its conventional sense, and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be employed is a DNA plasmid, the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding a fusion polypeptide within an expression cassette. Examples of promoters that can be used include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. A promoter can also be a tissue specific promoter, such as a neuron specific promoter, which can be natural or synthetic. Exemplary neuron-specific promoters include but are not limited to the Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, neuron-specific enolase (NSE) promoter, and synapsin I with a minimal CMV sequence (SynI-minCMV) promoter (Radhiyanti, et al. Neur. Lett., 75:135956 (2021). Another example is Scn10a promoter45678892.142 ATTORNEY DOCKET NO. YU 8723 PCT which regulates expression in neurons that normally express Nav1.8 channels (Lu VB et al J Neuroscience 201535(20):8021-34). b. Other Expression Control Elements The disclosed nucleic acids, including vectors, can include additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and / or improve transcriptional-translational coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. A polyadenylation signal is typically located downstream of the coding sequence for a fusion polypeptide or other therapeutic polypeptide within an expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene. An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding a fusion polypeptide or other therapeutic polypeptide, but downstream of a promoter sequence within an expression cassette of the vector. Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human beta-globin polyadenylation signal. Preferably, a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal. Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are not limited to, Woodchuck HBV Post- transcriptional regulatory element (WPRE), intron / exon sequence derived from human apolipoprotein A1 precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit beta- globin intron, or any combination thereof. Preferably, an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit beta-globin intron, and a splicing enhancer, which is referred to herein as “a triple enhancer sequence.” A vector, such as a DNA plasmid, can also include a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding the fusion45678892.143 ATTORNEY DOCKET NO. YU 8723 PCT polypeptide or other therapeutic polypeptide, or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColE1, pMB1, pUC, pSC101, R6K, and 15A, preferably pUC. Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., the disclosed fusion polypeptides and variants thereof. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr), and tetracycline resistance gene (Tetr), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc. An expression vector can include a tag sequence, such as those discussed above. 4. Host Cells Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell) can be used to, for example, produce the fusion polypeptides described herein. C. Delivery Vehicles Any of the disclosed compositions including, but not limited to polypeptides and / or nucleic acids, can be delivered to target cells using a delivery vehicle. The delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc. Delivery vehicles may be microparticles or nanoparticles. Nanoparticles are often utilized for intertissue application, penetration of cells, and certain routes of administration. The45678892.144 ATTORNEY DOCKET NO. YU 8723 PCT nanoparticles may have any desired size for the intended use. The nanoparticles may have any diameter from 10 nm up to about 1,000 nm. The nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In some embodiments the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm. Thus, in some embodiments, the delivery vehicles are nanoscale compositions, for example, 10 nm up to, but not including, about 1 micron. However, it will be appreciated that in some embodiments, and for some uses, the particles can be smaller, or larger (e.g., microparticles, etc.). Although many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it will be appreciated that in some embodiments and for some uses the carrier can be somewhat larger than nanoparticles. Such compositions can be referred to as microparticulate compositions. For example, a nanocarriers according to the present disclosure may be a microparticle. Microparticles can a diameter between, for example, 0.1 and 100 µm in size. 1. Polymers The delivery vehicle can be a particle containing one or more hydrophilic polymers. Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma- polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide); poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol), and copolymers thereof. The delivery vehicle can contain one or more hydrophobic polymers. Examples of suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3- hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes; poly(oxyethylene) / poly(oxypropylene)45678892.145 ATTORNEY DOCKET NO. YU 8723 PCT copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof. In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In some embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid). The particle can contain one or more biodegradable polymers. Biodegradable polymers can include polymers that are insoluble or sparingly soluble in water that are converted chemically or enzymatically in the body into water-soluble materials. Biodegradable polymers can include soluble polymers crosslinked by hydolyzable cross-linking groups to render the crosslinked polymer insoluble or sparingly soluble in water. Biodegradable polymers in the particle can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy- propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene and polyvinylpryrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. Exemplary biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. The particles can contain one or more amphiphilic polymers. Amphiphilic polymers can be polymers containing a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can contain one or more of the hydrophobic polymers above or a derivative or copolymer thereof. The hydrophilic polymer block can contain one or more of the45678892.146 ATTORNEY DOCKET NO. YU 8723 PCT hydrophilic polymers above or a derivative or copolymer thereof. In some embodiments the amphiphilic polymer is a di-block polymer containing a hydrophobic end formed from a hydrophobic polymer and a hydrophilic end formed of a hydrophilic polymer. In some embodiments, a moiety can be attached to the hydrophobic end, to the hydrophilic end, or both. In some embodiments, the particles contain a first amphiphilic polymer having a hydrophobic polymer block, a hydrophilic polymer block, and targeting moiety conjugated to the hydrophilic polymer block; and a second amphiphilic polymer having a hydrophobic polymer block and a hydrophilic polymer block but without the targeting moiety. The hydrophobic polymer block of the first amphiphilic polymer and the hydrophobic polymer block of the second amphiphilic polymer may be the same or different. Likewise, the hydrophilic polymer block of the first amphiphilic polymer and the hydrophilic polymer block of the second amphiphilic polymer may be the same or different. In some embodiments the particle contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). The nanoparticles can contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as "PGA", and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA", and caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as "PCL"; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as "PLGA"; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA- PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers". In certain embodiments, the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker. Other polymers include PLGA- poly(ε- carbobenzoxyl-L-lysine) (PLL) (i.e., PLGA-PLL). The particles can also contain one or more polymer conjugates containing end-to-end linkages between the polymer and a targeting moiety or a detectable label. For example, a modified polymer can be a PLGA-PEG-peptide block polymer. The particles can contain one or a mixture of two or more polymers. The particles may contain other entities such as stabilizers, surfactants, or lipids. The particles may contain a first polymer having a targeting moiety and a second polymer not having the targeting moiety. By45678892.147 ATTORNEY DOCKET NO. YU 8723 PCT adjusting the ratio of the targeted and non-targeted polymers, the density of the targeting moiety on the exterior of the particle can be adjusted. The particles can contain an amphiphilic polymer having a hydrophobic end, a hydrophilic end, and a targeting moiety attached to the hydrophilic end. In some embodiments the amphiphilic macromolecule is a block copolymer having a hydrophobic polymer block, a hydrophilic polymer block covalently coupled to the hydrophobic polymer block, and a targeting moiety covalently coupled to the hydrophilic polymer block. For example, the amphiphilic polymer can have a conjugate having the structure A-B-X where A is a hydrophobic molecule or hydrophobic polymer, B is a hydrophilic molecule or hydrophilic polymer, and X is a targeting moiety. Exemplary amphiphilic polymers include those where A is a hydrophobic biodegradable polymer, B is PEG, and X is a targeting moiety that targets, binds. In some embodiments the nanoparticle contains a first amphiphilic polymer having the structure A-B-X as described above and a second amphiphilic polymer having the structure A-B, where A and B in the second amphiphilic macromolecule are chosen independently from the A and B in the first amphiphilic macromolecule, although they may be the same. 2. Liposomes and Micelles In some embodiments, the carrier is a liposome or micelle. Liposomes are spherical vesicles composed of concentric phospholipid bilayers separated by aqueous compartments. Liposomes can adhere to and form a molecular film on cellular surfaces. Structurally, liposomes are lipid vesicles composed of concentric phospholipid bilayers which enclose an aqueous interior (Gregoriadis, et al., Int. J. Pharm., 300, 125-302005; Gregoriadis and Ryman, Biochem. J., 124, 58P (1971)). Hydrophobic compounds associate with the lipid phase, while hydrophilic compounds associate with the aqueous phase. Liposomes include, for example, small unilamellar vesicles (SUVs) formed by a single lipid bilayer, large unilamellar vesicles (LANs), which are vesicles with relatively large particles formed by a single lipid bilayer, and multi-lamellar vesicles (MLVs), which are formed by multiple membrane layers. Thus, the liposomes can have either one or several aqueous compartments delineated by either one (unilamellar) or several (multilamellar) phospholipid bilayers (Sapra, et al., Curr. Drug Deliv., 2, 369-81 (2005)). Multilamellar liposomes have more lipid bilayers for hydrophobic therapeutic agents to associate with. Thus, potentially greater amounts of therapeutic agent are available within the liposome to reach the target cell. Liposomes have the ability to form a molecular film on cell and tissue surfaces. Clinical studies have proven the efficacy of liposomes as a topical healing agent (Dausch, et al., Klin Monatsbl Augenheilkd 223, 974-83 (2006); Lee, et al., Klin Monatsbl Augenheilkd 221, 825-3645678892.148 ATTORNEY DOCKET NO. YU 8723 PCT (2004)). Liposomes have also been used in ophthalmology to ameliorate keratitis, corneal transplant rejection, uveitis, endophthalmitis, and proliferative vitreoretinopathy (Ebrahim, et al., 2005; Li, et al., 2007). Liposomes have been widely studied as drug carriers for a variety of chemotherapeutic agents (approximately 25,000 scientific articles have been published on the subject) (Gregoriadis, N Engl J Med 295, 765-70 (1976); Gregoriadis, et al., Int. J. Pharm.300, 125-30 (2005)). More than ten liposomal and lipid-based formulations have been approved by regulatory authorities and many liposomal drugs are in preclinical development or in clinical trials (Barnes, Expert Opin Pharmacother 7, 607-15 (2006); Minko, et al., Anticancer Agents Med Chem 6, 537-52 (2006)). The safety data with respect to acute, subchronic, and chronic toxicity of liposomes has been assimilated from the vast clinical experience of using liposomes in the clinic for thousands of patients. Carriers such as liposomes and micelles can be formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2-distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholines, 1-acyl-2-acyl-sn-glycero-3- phosphocholines, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. In some embodiments, the liposomes contain a phosphaditylcholine (PC) head group, and optionally sphingomyelin. In another embodiment, the liposomes contain DPPC. In a further embodiment, the liposomes contain a neutral lipid, such as 1 ,2-dioleoylphosphatidylcholine (DOPC). In certain embodiments, the liposomes are generated from a single type of phospholipid. In some embodiments, the phospholipid has a phosphaditylcholine head group, and, can be, for45678892.149 ATTORNEY DOCKET NO. YU 8723 PCT example, sphingomyelin. The liposomes may include a sphingomyelin metabolite. Sphingomyelin metabolites used to formulate the liposomes include, without limitation, ceramide, sphingosine, or sphingosine 1-phosphate. The concentration of the sphingomyelin metabolites included in the lipids used to formulate the liposomes can range from about 0.1 mol % to about 10 mol %, or from about 2.0 mol % to about 5.0 mol %, or can be in a concentration of about 1.0 mol %. Suitable cationic lipids in the liposomes include, but are not limited to, N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[1-(2,3- dioloyloxy)propyl]-Ν,Ν-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2- dialkyloxy-3-dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N- (N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoro-acetate (DOSPA), β- alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diC14-amidine, N-ferf-butyl-N'- tetradecyl-3-tetradecylamino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D- glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N , N , N' , N'- tetramethyl- , N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1 ,4-butanediammonium iodide. In one embodiment, the cationic lipids can be 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)- imidazolinium chloride derivatives, for example, 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)- heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and 1-[2- (hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl- hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl- hydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl-45678892.150 ATTORNEY DOCKET NO. YU 8723 PCT hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DSRIE). The lipids may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleolylglyceryl phosphatidylethanolamine). The molar ratio of a first phospholipid, such as sphingomyelin, to second lipid can range from about 5:1 to about 1:1 or 3:1 to about 1:1, or from about 1.5:1 to about 1:1, or the molar ratio is about 1:1. In some embodiments, liposomes or micelles include phospholipids, cholesterols and nitrogen-containing lipids. Examples include phospholipids, including natural phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean lecithin, and lysolecithin, as well as hydrogenated products thereof obtained in a standard manner. It is also possible to use synthetic phospholipids such as dicetyl phosphate, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine as well as homo- poly{N'--[N-(2-aminoethyl)-2-aminoethyl]aspartamide} P[Asp(DET)] and block-catiomer poly(ethyleneglycol) (PEG)-b-P[Asp(DET)]. In some embodiments, the liposomes are long circulating liposomes or stealth liposomes such as those reviewed in Immordino, et al, Int J Nanomedicine, 1(3):297–315 (2006)), which is specifically incorporated by reference herein in its entirety. For example, liposomes have been developed with surfaces modified with a variety of molecules including glycolipids and sialic acid. Long-circulating liposomes can include, for example, synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition. The PEG on the surface of the liposomal carrier can extend blood-circulation time while reducing mononuclear phagocyte system uptake (stealth liposomes) and serve as an anchor for the targeting moiety. Antibodies and antibody fragments are widely employed for targeting moieties for liposomes due to the high specificity for their target antigens. Referred to immunoliposomes, methods of generated targeted liposomes by coupling of antibodies to the liposomal surface are known in the art. Such techniques include, but are not limited to, conventional coupling and maleimide based techniques. See, for example, (Paszko and Senge, Curr Med Chem., 19(31):5239-77 (2012), Kelly, et al., Journal of Drug Delivery, Volume 2011 (2011), Article ID 727241, 11 pages).45678892.151 ATTORNEY DOCKET NO. YU 8723 PCT The micelles can be polymer micelles, for example, those composed of amphiphilic di-or tri-block copolymers made of solvophilic and solvophobic blocks (see, e.g., Croy and Kwon, Curr Pharm Des., 12(36):4669-84 (2006)). III. METHODS OF MAKING A. Methods for Producing fusion polypeptides Fusion proteins of Formula I, fragments thereof, or variants thereof, can be obtained, for example, by chemical synthesis, and more preferably, by recombinant production in a host cell. To recombinantly produce a fusion protein of Formula I, a nucleic acid containing a nucleotide sequence encoding the polypeptide can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding a fusion proteins of Formula I. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked. The nucleotide sequences encoding the fusion protein are usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the fusion protein is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA or may contain elements of both. The term, “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fusion protein. Expression vectors for use in expressing the fusion protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Expression vectors for use in expressing the fusion protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or45678892.152 ATTORNEY DOCKET NO. YU 8723 PCT heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol.1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809- 814), the CMV promoter (Boshart et al., Cell 41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells. In eukaryotic host cells, a number of viral-based expression systems can be utilized to express fusion proteins of Formula I. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors. Mammalian cell lines that stably express variant costimulatory polypeptides can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of polypeptides in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. Alternatively, the disclosed fusion polypeptides can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate. The expressed fusion proteins produced by the cells may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, releasing the fusion protein by mechanical cell disruption, such as ultrasonication or pressure, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate. After sonication a suitable concentration of NaCl can be added to further decrease the ability of host cell contaminants to bind to the cation exchange matrix. After cation-exchange chromatography the fusion protein may be eluted in a salt gradient and eluate fractions containing the fusion protein are collected. In some preferred45678892.153 ATTORNEY DOCKET NO. YU 8723 PCT forms, fusion protein is captured from lysate through its His tag. So IMAC (immobilized metal affinity chromatography) was used and then, after concentration of protein-containing fractions, they are subjected to size exclusion chromatography (SEC) for final purification. In some forms the fusion protein is purified from the periplasmic space, where the host cell is bacteria, for example, E. coli. This would include (1) centrifugation, (2) osmotic shock to release the protein from the cell wall compartment, (3) IMAC (Immobilized Metal Ion Affinity Chromatography), (4) SEC (Size Exclusion Chromatography). Disclosed fusion polypeptides can be isolated using, for example, chromatographic methods such as DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. For example, polypeptides in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein G column. As discussed above, in some embodiments, polypeptides can be “engineered” to contain an amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify the polypeptides. Methods for introducing random mutations to produce variant polypeptides are known in the art. Random peptide display libraries can be used to screen for polypeptides that interact with NNT. Techniques for creating and screening such peptide display libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No.4,946,778; Ladner et al., U.S. Pat. No.5,403,484 and Ladner et al., U.S. Pat. No.5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially. Polypeptide Conjugates Additionally, or alternatively, disclosed fusion polypeptides can be prepared as protein conjugates with one or more functional elements (e.g., protein transduction domains, fusogenic peptides, targeting molecules, tags, etc. chemically conjugated thereto. Methods for attaching peptides, small molecules, and other compounds to polypeptides are well known in the art and can include use of bifunctional chemical linkers such as N-succinimidyl (4-iodoacetyl)- aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate; 4-succinimidyl-oxycarbonyl- ^-(2-pyridyldithio) toluene; sulfosuccinimidyl-6-[alpha-methyl- ^-(pyridyldithiol)-toluami-do] hexanoate; N-succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3 (-(-2- pyridyldithio)-proprionamido] hexanoate; sulfosuccinimidyl-6-[3 (-(-2-pyridyldithio)- propionamido] hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,45678892.154 ATTORNEY DOCKET NO. YU 8723 PCT dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like. Further bifunctional linking molecules are discussed in, for example, U.S. Pat. Nos.5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877. The linker can be cleavable or noncleavable. Highly stable linkers can reduce the amount of payload that falls off in circulation, thus improving the safety profile, and ensuring that more of the payload arrives at the target cell. Linkers can be based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the active agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials (see, e.g., Brentuximab vedotin which includes an enzyme-sensitive linker cleavable by cathepsin; and Trastuzumab emtansine, which includes a stable, non-cleavable linker). In particular embodiments, the linker is a peptide linker cleavable by Edman degradation (Bąchor, et al., Molecular diversity, 17 (3): 605–11 (2013)). B. Methods for Producing Isolated Nucleic Acid Molecules Isolated nucleic acid molecules encoding polypeptides such as the disclosed fusion polypeptides and other therapeutic proteins can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant costimulatory polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293. Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoroamidite technology for45678892.155 ATTORNEY DOCKET NO. YU 8723 PCT automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Polypeptide encoding nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992. Examples of amino acid positions that can be modified include those described herein. C. Methods of Making Delivery Vehicles 1. Particle Formation Methods of making delivery vehicles are known in the art. See, e.g., U.S. Published Application No.2019 / 0330317, which is specifically incorporated by reference herein in its entirety. For example, in some embodiments, a particle is prepared using an emulsion solvent evaporation method. For example, a polymeric material is dissolved in a water immiscible organic solvent and mixed with a drug solution or a combination of drug solutions. In some embodiments the polymer solution contains one or more polymer conjugates as described above. In another embodiment, a multimodal nanoparticle is prepared using nanoprecipitation methods or microfluidic devices. Polymeric material is mixed with a drug or drug combinations in a water miscible organic solvent. Methods of making nanoparticles using microfluidics are known in the art. Suitable methods include those described in U.S. Published Application No. 2010 / 0022680 A1 by Karnik et al. Other methods of making particles include, but are not limited to, solvent evaporation, hot melt microencapsulation, solvent removal, spray-drying, and hydrogel particles formation. Methods of manufacturing liposomes are known in the art and can include, for example, drying down of the lipids from organic solvents, dispersion of the lipids in aqueous media, purification of the resultant liposomes, and analysis of the final product. Some methods of liposome manufacture include, for example, extrusion methods, the Mozafari method, the polyol dilution method, the bubble method, and the heating method. The micelles may be prepared in a conventional manner, for example, by reversed-phase evaporation, ether injection, surfactant-based techniques, etc. Polymer micelle formulations45678892.156 ATTORNEY DOCKET NO. YU 8723 PCT utilizing a block copolymer having a hydrophilic segment and a hydrophobic segment have been disclosed, e.g., in U.S. Application No.2016 / 0114058, WO 2009 / 142326 A1 and WO 2010 / 013836 A1. 2. Methods of Encapsulating or Attaching Molecules to Particles Delivery vehicles can be used to deliver the disclosed compositions. For example, a fusion polypeptide as disclosed herein or other therapeutic protein, with or without a heterologous sequence, or a nucleic acid encoding the same can be encapsulated in the delivery vehicle. Additionally, or alternatively, the fusion polypeptide and / or nucleic acid can be conjugated to one or more elements of the delivery vehicle. In addition, or alternative to any of the foregoing, the delivery vehicle can include one or more functional elements, such as protein transduction domains, fusogenic peptides, targeting molecules, etc., can be encapsulated or more preferably conjugated, most preferably exterior surface conjugated, to the delivery vehicle. These can be coupled using standard techniques. The targeting molecule or therapeutic molecule to be delivered can be coupled directly to the polymer or to a material such as a fatty acid which is incorporated into the polymer. Functionality refers to conjugation of a ligand to the surface of the particle via a functional chemical group (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the surface of the particle and present on the ligand to be attached. Functionality may be introduced into the particles in two ways. The first is during the preparation of the particles, for example during the emulsion preparation of particles by incorporation of stabilizers with functional chemical groups. A second is post-particle preparation, by direct crosslinking particles and ligands with homo- or heterobifunctional crosslinkers. This second procedure may use a suitable chemistry and a class of crosslinkers (CDI, EDAC, glutaraldehydes, etc. as discussed in more detail below) or any other crosslinker which couples ligands to the particle surface via chemical modification of the particle surface after preparation. This second class also includes a process whereby amphiphilic molecules such as fatty acids, lipids or functional stabilizers may be passively adsorbed and adhered to the particle surface, thereby introducing functional end groups for tethering to ligands. Methods of polymer synthesis are described, for instance, in Braun et al. (2005) Polymer Synthesis: Theory and Practice. New York, NY: Springer. The polymers may be synthesized via step-growth polymerization, chain-growth polymerization, or plasma polymerization. In some embodiments an amphiphilic polymer is synthesized starting from a hydrophobic polymer terminated with a first reactive coupling group and a hydrophilic polymer terminated45678892.157 ATTORNEY DOCKET NO. YU 8723 PCT with a second reactive coupling group capable of reacting with the first reactive coupling group to form a covalent bond. One of either the first reactive coupling group or the second reactive coupling group can be a primary amine, where the other reactive coupling group can be an amine-reactive linking group such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. One of either the first reactive coupling group or the second reactive coupling group can be an aldehyde, where the other reactive coupling group can be an aldehyde reactive linking group such as hydrazides, alkoxyamines, and primary amines. One of either the first reactive coupling group or the second reactive coupling group can be thiol, where the other reactive coupling group can be a sulfhydryl reactive group such as maleimides, haloacetyls, and pyridyl disulfides. In some embodiments a hydrophobic polymer terminated with an amine, or an amine- reactive linking group is coupled to a hydrophilic polymer terminated with complimentary reactive linking group. For example, an NHS ester activated PLGA can be formed by reacting PLGA-CO(OH) with NHS and a coupling reagent such as dicyclohexylcarbodiimide (DCC) or ethyl(dimethylaminopropyl) carbodiimide (EDC). The NHS ester activated PLGA can be reacted with a hydrophilic polymer terminated with a primary amine, such as a PEG-NH2 to form an amphiphilic PLGA-b-PEG block copolymer. In some embodiments a conjugate of an amphiphilic polymer with a functional moiety is formed using the same or similar coupling reactions. In some embodiments the conjugate is made starting from a hydrophilic polymer terminated on one end with a first reactive coupling group and terminated on a second end with a protective group. The hydrophilic polymer reacts with a targeting moiety having a reactive group that is complimentary to the first reactive group to form a covalent bond between the hydrophilic polymer and the targeting moiety. The protective group can then be removed to provide a second reactive coupling group, for example to allow coupling of a hydrophobic polymer block to the conjugate of the hydrophilic polymer with the targeting moiety. A hydrophobic polymer terminated with a reactive coupling group complimentary to the second reactive coupling group can then be covalently coupled to form the conjugate. Of course, the steps could also be performed in reverse order, i.e., a conjugate of a hydrophobic polymer and a hydrophilic polymer could be formed first followed by deprotection and coupling of the targeting moiety to the hydrophilic polymer block. In some embodiments a conjugate is formed having a moiety conjugated to both ends of the amphiphilic polymer. For example, an amphiphilic polymer having a hydrophobic polymer block, and a hydrophilic polymer block may have targeting moiety conjugated to the hydrophilic45678892.158 ATTORNEY DOCKET NO. YU 8723 PCT polymer block and an additional moiety conjugated to the hydrophobic polymer block. In some embodiments the additional moiety can be a detectable label. In some embodiments the additional moiety is a therapeutic, prophylactic, or diagnostic agent. For example, additional moiety could be a moiety used for radiotherapy. The conjugate can be prepared starting from a hydrophobic polymer having on one end a first reactive coupling group and another end first protective group and a hydrophilic polymer having on one end a second reactive coupling group and on another end a second protective group. The hydrophobic polymer can be reacted with the additional moiety having a reactive coupling group complimentary to the first reactive coupling group, thereby forming a conjugate of the hydrophobic polymer to the additional moiety. The hydrophilic polymer can be reacted with a targeting moiety having a reactive coupling group complimentary to the second reactive coupling group, thereby forming a conjugate of the hydrophilic polymer to the targeting moiety. The first protective group and the second protective group can be removed to yield a pair of complimentary reactive coupling groups that can be reacted to covalently link the hydrophobic polymer block to the hydrophilic polymer block. IV. Pharmaceutical Compositions The disclosed compositions alone or in a delivery vehicle can be formulated with appropriate pharmaceutically acceptable carriers into pharmaceutical compositions for administration to an individual in need thereof. The formulations can be administered enterally (e.g., oral) or parenterally (e.g., by injection or infusion). The disclosed compositions can be formulated for parenteral administration. “Parenteral administration”, as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesional, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intrathecally, intraumbilically, by injection, and by infusion. In some embodiments, the disclosed compositions are administered systemically by, for example, injection or infusion. In some embodiments, the compositions are administered locally by injection or infusion. For example, in more specific embodiments, the compositions are administered to sensory neurons in dorsal root ganglion (DRG) (e.g., by injection or infusion, intrathecally to reach the tissues where sensory neurons reside.), or to the central nervous system, particularly the brain, by convection enhanced delivery (CED).45678892.159 ATTORNEY DOCKET NO. YU 8723 PCT Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required nanocarrier size in the case of dispersion and / or by the use of surfactants. In many cases, isotonic agents, for example, sugars or sodium chloride are included Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof. Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer®401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.45678892.160 ATTORNEY DOCKET NO. YU 8723 PCT The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s). The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol. Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The powders can be prepared in such a manner that the nanocarriers are porous in nature, which can increase dissolution of the nanocarriers. Methods for making porous nanocarriers are well known in the art. Enteral formulations are prepared using pharmaceutically acceptable carriers. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in the dosage form include hydrophobic or hydrophilic polymers and pH dependent or independent polymers. Exemplary hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins. Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.45678892.161 ATTORNEY DOCKET NO. YU 8723 PCT Controlled release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington – The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA). The disclosed compounds alone or in a particle formulation can also be applied topically. Compositions and methods for topical administration of nucleic acids are known in the art (Gurevich, et al., Nature Medicine 28:780–788 (2022), disclosing topical nucleic acid administration using herpes simplex virus type 1 (HSV-1) vector; Sheridan et al., Nature Biotechnology 40: 809–811 (2022)). Nucleic acid constructs encoding disclosed pain active agents can be expressed for topical delivery using vectors such as herpes simplex virus type 1 (HSV-1) vector. Topical administration can include application to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In some embodiments, the compositions are administered in combination with transdermal or mucosal transport elements. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.45678892.162 ATTORNEY DOCKET NO. YU 8723 PCT Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules, or lozenges. Oral formulations may include excipients or other modifications to the particle which can confer enteric protection or enhanced delivery through the GI tract, including the intestinal epithelia and mucosa (see Samstein, et al., Biomaterials, 29(6):703-8 (2008). Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, and transdermal patches. The formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The compounds can also be formulated for intranasal delivery, pulmonary delivery, or inhalation. The compositions may further contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, buffers, and combination thereof. In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For topical applications, repeated application can be done, or a patch can be used to provide continuous administration of the compounds over an extended period of time “Buffers” are used to control the pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine. “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4thEd., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate. “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose,45678892.163 ATTORNEY DOCKET NO. YU 8723 PCT hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate. “Penetration enhancers” are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art. “Preservatives” can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal. “Surfactants” are surface- active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non- ionic surfactant is stearyl alcohol. (a) Emulsions An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. In particular embodiments, the non-miscible components of the emulsion include a lipophilic component and an aqueous component. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous45678892.164 ATTORNEY DOCKET NO. YU 8723 PCT substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers. The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers. A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) comprised of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes. (b) Lotions A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface.45678892.165 ATTORNEY DOCKET NO. YU 8723 PCT (c) Creams Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments, as they are generally easier to spread and easier to remove. The difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the water- base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %. (d) Ointments Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components. (e) Gels Gels are semisolid systems containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.45678892.166 ATTORNEY DOCKET NO. YU 8723 PCT (f) Foams Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use. In one embodiment, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation. The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery. Pulmonary administration of therapeutic compositions including low molecular weight drugs has been observed, for example, beta-androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the sub epithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.45678892.167 ATTORNEY DOCKET NO. YU 8723 PCT Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration. Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. Solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension. In one embodiment, compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, "minor amounts" means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.45678892.168 ATTORNEY DOCKET NO. YU 8723 PCT Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA). Dry powder formulations ("DPFs") with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large "carrier" particles (containing no drug) have been co- delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits. Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent. The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different EGS may be administered to target different regions of the lung in one administration. Formulations for pulmonary delivery include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Formulations and methods of making such formulations containing nucleic acid are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, the liposome can include a ligand molecule specific for a receptor on the surface of the target cell to direct the liposome to the target cell.45678892.169 ATTORNEY DOCKET NO. YU 8723 PCT IV. METHODS OF USE Methods of treatment are provided and typically include administering a subject in need thereof an effective amount of a disclosed fusion polypeptide or nucleic acid, optionally, but preferably, in a pharmaceutical composition. Pain can be anything from a slightly bothersome, such as a mild headache, to something excruciating and emergent, such as the chest pain that accompanies a heart attack, or pain of kidney stones. Pain can be acute, meaning new, subacute, lasting for a few weeks or months, and chronic, when it lasts for more than 3 months. Acute pain may come from inflammation, tissue damage, injury, illness, or recent surgery. It usually lasts less than a week or two. The pain usually ends after the underlying cause is treated or has been resolved. Chronic or persistent pain is pain that carries on for longer than 12 weeks despite medication or treatment. Thus, in some forms, subject is suffering from chronic pain. Chronic pain can affect people living with diabetes, arthritis, fibromyalgia, irritable bowel syndrome, back pain, etc. Cancer pain affects most people with advanced cancer. Headaches affect millions of U.S. adults. Some of the most common types of chronic headaches are migraines, cluster headaches, and tension headaches. In some forms, the subject is a human. However, the subject can be a mammal, such as a primate, a domestic animal, zoo animal, including but not limited to dogs, cats, chimpanzee, gorilla, etc. The methods include delivering / administering an effective amount of the disclosed pain active agents to the subject. In some forms the methods include delivering pain active agents in the form of a polypeptide. In some forms, the method include delivering a vector encoding a pain active agent to tissue / cells in the subject, such as sensory neurons. In some forms, delivering pain active agents to tissues in living animals depends on introducing genetic material encoding pain active agents to peripheral sensory neurons. One approach to this delivery is expressing a pain active agent in adeno associated virus (AAV) capsid which can be delivered intrathecally to reach the tissues where sensory neurons reside. In some forms the vector encoding one or more pain active agents is administered as a topical formulation. The disclosed compositions and methods can be further understood by means of the following non-limiting paragraphs. Paragraph 1. A fusion polypeptide or a functional fragment or variant thereof and an optional heterologous sequence, optionally packaged in or otherwise associated with a delivery vehicle, wherein the fusion polypeptide is represented by the Formula I, D1-L1-D2,45678892.170 ATTORNEY DOCKET NO. YU 8723 PCT where D1 is a first bioactive domain, D2 is a second bioactive domain, and L1 is an optional flexible linker, wherein the first or second bioactive domains comprise a domain with selective binding to Nav1.8 channels and domain with either ubiquitin ligase activity, ubiquitin ligase recruitment activity or lysosome recruitment activity. Paragraph 2. The polypeptide of paragraph 1, wherein domain with ubiquitin ligase activity comprises HECT (homologous to the E6-AP C-terminus) of NEDD4 and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1). Paragraph 3. The polypeptide of paragraph 1, wherein the domain with ubiquitin ligase activity comprises a RING (Really Interesting New Gene) domain and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1). Paragraph 4. The polypeptide of paragraph 1, wherein the domain with ubiquitin ligase recruitment activity comprises a degron peptide and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1). Paragraph 5. The polypeptide of paragraph 1, wherein the fusion poplypeptide includes a lysosome recruiting domain and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1). Paragraph 6. The polypeptide of paragraph 1, wherein the domain with lysosome recruitment domain comprises SEQ ID NO: 35 or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35 and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1). Paragraph 7. The polypeptide of any one of paragraphs 1-6, wherein D1 or D2 comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,. Paragraph 8. The polypeptide of any one of paragraphs 1-7, wherein D1 or D2 comprises SEQ ID NO:5 or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5. Paragraph 9. The polypeptide of any one of paragraphs 1-8, comprising SEQ ID NO:2 or 3 and / or SEQ ID NO: 5.45678892.171 ATTORNEY DOCKET NO. YU 8723 PCT Paragraph 10. The polypeptide of any one of paragraphs 1-8, comprising SEQ ID NO:26 and / or SEQ ID NO:5. Paragraph 11. The polypeptide of any one of paragraphs 1-8, comprising SEQ ID NO:27 and / or SEQ ID NO:5. Paragraph 12. The polypeptide of any one of paragraphs 1-8, comprising SEQ ID NO:28 and / or SEQ ID NO:5. Paragraph 13. The polypeptide of any one of paragraphs 1-8, comprising SEQ ID NO:29 and / or SEQ ID NO:5. Paragraph 14. The polypeptide of any one of paragraphs 1-13, wherein L1 is selected from the group consisting of SEQ ID NO: 7-12. Paragraph 15. The polypeptide of any one of paragraphs 1-14, wherein D1 comprises SEQ ID NO:2 and D2 comprises SEQ ID NO:5. Paragraph 16. The polypeptide of any one of paragraphs 1-14, wherein D1 comprises SEQ ID NO:26 and D2 comprises SEQ ID NO:5. Paragraph 17. The polypeptide of any one of paragraphs 1-14, wherein D1 comprises SEQ ID NO:27 and D2 comprises SEQ ID NO:5. Paragraph 18. The polypeptide of any one of paragraphs 1-14, wherein D1 comprises SEQ ID NO:28 and D2 comprises SEQ ID NO:5. Paragraph 19. The polypeptide of any one of paragraphs 1-14, wherein D1 comprises SEQ ID NO:29 and D2 comprises SEQ ID NO:5. Paragraph 20. The polypeptide of any one of paragraphs 1-19, comprising a heterologous sequence, wherein the heterologous sequence comprises one or more of a protein transduction domain, fusogenic polypeptide, targeting signal, expression and / or purification tag. Paragraph 21. The polypeptide of any one of paragraphs 1-20, comprising a delivery agent. Paragraph 22. The polypeptide of any one of paragraphs 1-21, wherein the polypeptide can interact with selective binding to Nav1.8 channels. Paragraph 23. The polypeptide of any one of paragraphs 1-20, comprising a mutated PEST motif with reduce activity. Paragraph 24. The polypeptide of any one of paragraphs 1-23, wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5. Paragraph 25. The polypeptide of any one of paragraphs 1-24, wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO 2.45678892.172 ATTORNEY DOCKET NO. YU 8723 PCT Paragraph 26. The polypeptide of any one of paragraphs 1-25 wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3. Paragraph 27. A nucleic acid comprising a nucleic acid encoding the polypeptide of any one of paragraphs 1-26, optionally packaged in a delivery vehicle. Paragraph 28. The nucleic acid of paragraph 27 comprising SEQ ID NO:1. Paragraph 29. The nucleic acid of any one of paragraphs 27 or 28, wherein the nucleic acid is RNA or DNA. Paragraph 30. The nucleic acid of any one of paragraphs 27-29, wherein the nucleic acid comprises an expression control sequence(s). Paragraph 31. The nucleic acid of any one of paragraphs 27-30, wherein the nucleic acid is a vector. Paragraph 32. The nucleic acid of paragraph 31, wherein the nucleic acid is a viral vector. Paragraph 32. The nucleic acid of any one of paragraphs 27-30, wherein the nucleic acid is mRNA. Paragraph 33. The nucleic acid of any one of paragraphs 27-32, wherein the nucleic acid comprises a promotor. Paragraph 34. The nucleic acid of paragraph 33, wherein the promotor is a neuron-specific promoter. Paragraph 35. The nucleic acid of paragraph 34, wherein the neuron-specific promoter is selected from the group consisting of t Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, neuron-specific enolase (NSE) promoter, and synapsin I with a minimal CMV sequence (SynI-minCMV) promoter. Paragraph 36. The nucleic acid of any one of paragraphs 27-35 comprising one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto. Paragraph 37. The nucleic acid of any one of paragraphs 32-36, wherein the viral vector is an Adeno-associated virus (AAV)- Paragraph 38. The nucleic acid of any one of paragraphs 27-37 comprising the delivery vehicle. Paragraph 39. The polypeptide of any one of paragraphs 1-26 or nucleic acid of any one of paragraphs 27-38, wherein the delivery vehicle is formed of polymeric particles, inorganic particles, silica particles, liposomes, micelles, or multilamellar vesicles, optionally wherein the delivery vehicles comprise one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.45678892.173 ATTORNEY DOCKET NO. YU 8723 PCT Paragraph 40. A pharmaceutical composition comprising the polypeptide of any one of paragraphs 1-26 or nucleic acid of any one of paragraphs 27-38 alone or packaged in a delivery vehicle optionally formed from formed of polymeric particles, inorganic particles, silica particles, liposomes, micelles, or multilamellar vesicles, optionally wherein the delivery vehicles comprise one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto. Paragraph 41. A method of treating a subject in need thereof comprising administering the subject an effective amount of the pharmaceutical composition of paragraph 40. Paragraph 42. The method of paragraph 41, wherein the subject has acute pain. Paragraph 43. The method of paragraph 42, wherein the subject has chronic pain Paragraph 44. The method of any one of paragraphs 41-43, wherein the pain results from a condition selected from the group consisting of diabetes, arthritis, fibromyalgia, irritable bowel syndrome, back pain, surgery, cancer, migraines, neurogenic pain, and orchialgia. Paragraph 45. The method of any one of paragraphs 41-44, wherein the pharmaceutical composition comprises the fusion protein of Formula I. Paragraph 46. The method of any of any one of paragraphs 41-44, wherein the pharmaceutical composition comprises a nucleic acid encoding the fusion protein of Formula I. Paragraph 47. The method of any of any one of paragraphs 41-44, wherein the pharmaceutical composition comprises a nucleic acid encoding the fusion protein of Formula I, in a vector. Paragraph 48. The method of paragraph 45, wherein the vector is an adeno associated virus (AAV) capsid. Paragraph 49. The method of paragraph 48, wherein the AAV capsid is selected from the group consisting of AAV1, AAV4, AAV5, AAV6, AAV8, and AAV9. Paragraph 50. The method of any one of paragraphs 41-49, wherein the pharmaceutical composition is delivered intrathecally. Paragraph 51. The method of any one of paragraphs 41-50, wherein the pharmaceutical composition is delivered via injection or infusion Paragraph 52. The method of one of paragraphs 41-51, wherein the pharmaceutical composition is effective to reduce expression of Nav1.8 channels in the subject.45678892.174 ATTORNEY DOCKET NO. YU 8723 PCT Examples Methods Electrophysiology Rodent DRG (dorsal root ganglion) Neuron Isolation Animals used in this study were 2-4 d old Sprague-Dawley rat pups or 4–6-week-old NaV1.8-null mice. DRGs from these animals were harvested and dissociated as described previously1,2. In every culture, neurons were harvested from one female and one male animal so that the number of neurons from each sex were randomly distributed. Briefly, dissected DRGs were incubated at 37° C for 20 min in complete saline solution (CSS) [in mM: 137 NaCl, 5.3 KCl, 1 MgCl2, 25 sorbitol, 3 CaCl2, and 10 HEPES (pH 7.2), adjusted with NaOH], supplemented with 0.6 EDTA and collagenase A (1.5 mg / ml). DRG tissue was then incubated for 20 min at 37° C in CSS containing collagenase D (1.5 mg / mL;), 0.6 EDTA, and papain (30 U / ml). DRG tissue was centrifuged and triturated in 1 ml of DRG culture medium DMEM / F12 (1:1) with penicillin (100 U / ml), streptomycin (0.1 mg / ml), 2 L-glutamine, and 10% FBS containing BSA (1.5 mg / ml) and trypsin inhibitor (1.5 mg / ml). The suspended neurons were then filtered through a 70 μm nylon mesh cell strainer, and then washed with DRG culture medium. Rodent SCG (superior cervical ganglion) Neuron Isolation SCG neurons were isolated from neonatal (birth to 5-day-old) Sprague–Dawley rats as described previously (Han, et al. Neuromolecular Med 2015 Vol.17 Issue 2 Pages 158-69. PMID: 25791876). Briefly, SCGs were harvested, incubated at 37°C for 20 min in oxygenated complete saline solution (CSS) (in mM: 137 NaCl, 5.3 KCl, 1 MgCl2, 25 sorbitol, 3 CaCl2, and 10 HEPES, adjusted to pH 7.2 with NaOH) containing 1.5 mg / ml Collagenase A (Roche Diagnostics) and 0.6 mM EDTA and then exchanged with an oxygenated, 37°C CSS solution containing 1.5 mg / ml Collagenase D (Roche), 0.6 mM EDTA, and 30 U / ml papain (Worthington Biochemicals) for another 20 min. Tissue was then centrifuged and triturated in SCG media: DMEM / F12 (1:1) with 100 U / ml penicillin, 0.1 mg / ml streptomycin (Invitrogen) and 10% fetal bovine serum (Hyclone), which contained 1.5 mg / ml bovine serum albumin (Sigma-Aldrich) and 1.5 mg / ml trypsin inhibitor (Sigma). Electroporation -rat pup DRG neurons After the preparation of DRG neuron suspension, the cells were centrifuged for 3 min (100×g) and the supernatant was removed. Cells were gently resuspended at room temperature in 100 μL of a mixed Nucleofector solution (82 μL NucleofectorTM solution + 18 μL Supplement).45678892.175 ATTORNEY DOCKET NO. YU 8723 PCT Then, a 100 μL cell suspension with DNA (1.5 μg for all constructs except for GFP-control (0.3 ug)) was mixed by pipetting. Afterwards, a 20 μL cell suspension was transferred into a strip provided in the Nucleofector kit (Lonza; cat no. V4XP-3032). The sample was covered with the bottom of the strip, and air bubbles were avoided while pipetting. The strip was closed with the cap. The strip was inserted into its holder in the Nucleofector. Program DN-100 was used for rat DRG neurons. After transfection, 60 μL of pre-warmed BSA / TI solution was immediately added, and 80 μL were seeded per coverslip. DRG medium was added to a final volume of 1 mL per well 30 min after seeding, and the cells were incubated at 37º C in 95% O2 and 5% CO2. Electroporation – Nav1.8-null mouse neurons NaV1.8-null mouse neurons were transfected with a Nucleofector IIS and Amaxa Basic Neuron SCN Nucleofector Kit. Briefly, following harvest, the cell suspension was centrifuged (100 × g for 3 min), and the cell pellet was resuspended in 20 µL of Nucleofector solution, mixed with the following amounts of DNA depending on the experiment: 1.5 µg for human- NaV1.8, 1.5 µg for UbiquiNav, 1.5 µg for Sclt1, 1.5 µg for SCLT1, 0.1 µg mCherry. After electroporation using Nucleofector IIS and protocol SCN-BNP 6, 100 μL of calcium-free DMEM (37° C) was added, and cells were incubated at 37° C for 5 min to recover. The cell mixture was then diluted with DRG media containing 1.5 mg / mL BSA (low endotoxin) and 1.5 mg / mL trypsin inhibitor (Sigma), seeded 100 µl onto poly-D-lysine / laminin-coated coverslips (BD), and incubated at 37° C in a 95% air / 5% CO2 (vol / vol) incubator for 45 min to allow neurons to attach to the coverslips. After 45 min, 0.9 mL of DRG media was added into each well, and the DRG neurons were maintained at 37° C in a 95% air / 5% CO2 (vol / vol) incubator before voltage-clamp recordings. Electroporation – rat pup SCG neurons After trituration, the SCG cell suspension was transfected with WT or G699R mutant constructs with a Nucleofector IIS devices (Lonza), using the Amaxa®Basic Neuron SCN NucleofectorTMKit (VSPI-1003) and SCN Basic Neuro Protocol 6. Transfected neurons were allowed to recover for 5 min at 37°C in calcium-free DMEM media at 95% air / 5% CO2 (vol / vol) incubator. The cell suspension was diluted with SCG media containing 1.5 mg / ml bovine serum albumin and 1.5 mg / ml trypsin inhibitor, seeded onto poly-D-lysin / laminin precoated coverslips (BD Biosciences) and incubated at 37°C, 40 min to allow SCG neurons to attach to the coverslips. SCG media was then added, and the cells were maintained at 37°C in a 95% air / 5% CO2(vol / vol) incubator for 40 h before voltage-clamp recording.45678892.176 ATTORNEY DOCKET NO. YU 8723 PCT General Voltage-Clamp Recordings Patch pipettes were fabricated from borosilicate glass (World Precision Instruments) using a P-97 puller (Sutter Instruments) and fire-polished for a resistance of 0.8-1.2 megaohms when filled with internal solution. Unless otherwise noted, the pipette internal solution contained (in mM): 140 CsF, 10 NaCl, 1.1 EGTA, 10 HEPES, 20 Dextrose (pH 7.3 with CsOH, adjusted to 310 mOsm / L with dextrose). External bath solution contained (in mM): 140 NaCl, 20 TEA-Cl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 Sucrose, 0.1 CdCl2, ± 0.001 TTX (pH 7.3 with NaOH, adjusted to 320 mOsm / L with sucrose). Macroscopic currents were recorded in voltage-clamp mode using an EPC-10 amplifier and the PatchMaster Next program (HEKA Electronik). Sodium currents were recorded in the whole-cell configuration. Cells with a leak current >200 pA were excluded. Series resistance compensation of 80-90% was applied to reduce voltage error. Cells were excluded if the voltage error exceeded 5 mV. Recordings were sampled at 50 kHz through a low-pass Bessel filter of 10 kHz. After achieving the whole-cell configuration, a 5-min delay was applied to allow adequate time for the pipette solution and cytoplasmic milieu to equilibrate. Unless otherwise noted, Cells were held at -80 mV. I-V relationships generated by the voltage protocol were fit according to the following equation: ^ =^^^(^^^ ) ^^^^(^^.^^^)[Equation 1] ) from DRG neurons Transfected DRG neurons with diameters between 20-30 µM were selected for recording. Positively transfected neurons were identified by green (for eGFP linked constructs) or red (for mRuby or mCherry linked constructs) fluorescence. After achieving the whole-cell configuration, neurons were held at -80 mV. To isolate TTX-R NaV1.8 currents, 1 µM TTX was included in the bath. To evoke currents through NaV1.8, a standard activation protocol was used. Briefly, a series of 100ms step depolarizations from -80 to +40 mV in 5-mV were applied increments applied at 10s interpulse intervals. To investigate inactivation of NaV1.8, a standard inactivation protocol was used. Briefly, conditioning pulses of 500 ms duration ranging from - 140 to +30 mV in 10 mV increments were applied from a holding potential of -80 mV. Following the conditioning pulse, a 40 ms test pulse to +5 mV was applied to determine the fraction of sodium channels still available for opening.45678892.177 ATTORNEY DOCKET NO. YU 8723 PCT Recordings of human Nav1.8 currents in transfected Nav1.8-null neurons DRGs from adult male (4–6 week-old) NaV1.8-null mice were harvested and dissociated as described previously and transfected with relevant constructs via electroporation56. Positively transfected neurons were identified by green (for eGFP linked constructs) or red (for mRuby or mCherry linked constructs) fluorescence. To isolate TTX-R hNaV1.8 currents, 1 uM TTX was included in the bath. Voltage-clamp protocols described above were used to assess hNaV1.8 current density. Human DRG Neurons DRG neurons from a male 23-year-old donor were used for these experiments. The external bath solution for human DRG recordings contained (in mM): 30 NaCl, 110 Choline-Cl, 20 TEA-Cl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 Sucrose, 0.1 CdCl2, ± 0.001 TTX (pH 7.3 with NaOH, adjusted to 320 mOsm / L with sucrose). Even with only 30 Na+ in the bath, recordings from human DRG neurons were frequently affected by space clamp and voltage-error issues. A prepulse protocol (described previously3,4) to generate better space clamp in the human DRG recordings. Briefly, neurons were held at a potential of −80 mV (to inactivate non-NaV1.8 TTX-R channels). Then, patched cells were pre-pulsed to an individualized potential for 10 ms to inactivate axonal sodium currents. This prepulse potential was found to be the most negative voltage that could trigger an axonal spike without activating somatic channels – usually −35 to −25 mV. This was then followed by returning the cells to a hyperpolarized interpulse potential for 1 ms to allow for somatic but not axonal channel recovery. The interpulse potential was determined as the most negative potential that would not recover inadequately clamped sodium channels – usually between −100 and −120 mV. Finally, somatic sodium currents were triggered by subsequent 100 ms test pulses from −80 to +40 mV in 5 mV increments. TTX-S Current Recordings from DRG neurons Transfected DRG neurons between 20-30 mM were selected for recording. The internal pipette solution was the standard voltage clamp solution, but the external solution did not contain TTX. After achieving the whole-cell configuration, neurons were held at -90 mV (a potential at which most TTX-S channels are not inactivated). Then, to record total sodium currents, a series of 100ms step depolarizations from -100 to +40 mV in 5-mV increments were applied at 10s interpulse intervals. After this set of data was recorded, the holding potential was switched to -50 mV (a potential at which predominantly NaV1.8 channels are active) and the same voltage protocol was applied to the cell. The TTX-S component of the total sodium current was determined by reference subtraction of the recordings at -50 mV from those at -90 mV. IV45678892.178 ATTORNEY DOCKET NO. YU 8723 PCT curves were plotted and fit with Equation 1. Peak current density was defined as previously described. Voltage-clamp recordings of DRG neurons with iUbiquiNav DRG neurons were transfected with MTD-PYR1-P2A-mRuby2-P2A-HECT-ABI1 (iUbiquiNav) or GFP control and identified by appropriate fluorescence. Since reducing current rundown was paramount to the success of these experiments, several steps were taken to prefer neuronal health while in the whole-cell configuration. The extracellular solution for these experiments was DMEM without phenol red (Composition: 1.1 CaCl2, 0.0000052 CuSO4, 0.000124 Fe(NO3)3, 0.0015 FeSO4, 0.3 MgCl2, 0.4 MgSO4, 4.2 KCl, 29 NaHCO3, 120.6 NaCl, 0.5 Na2HPO4, 0.45 NaH2PO4, 0.0015 ZnSO4, 0.5 sodium pyruvate, and 17.5 glucose supplemented with 15 mM HEPES, adjusted with dextrose to 305 mOsm). The intracellular solution was (in mM): 140 CsF, 10 CsCl, 5 EGTA, 10 HEPES, 2 Na2ATP, 0.3 Na3GTP, 10 Dextrose, 2.5 mM Na2Phosphocreatine, pH 7.3 with CsOH, 310 mOsm). After allowing 5 minutes to pass in the whole cell configuration, voltage-dependence of NaV1.8 activation was assessed as described above. Then, a voltage protocol which applied 100 ms step voltages to the peak of channel activation every 10 seconds was started and continued for 1 hour. At sweep 5, perfusion of 1 uM Mandipropamid commenced and was continued until 10 mL of fluid had been exchanged (20x bath volume). A post-perfusion I-V curve was then captured. Fluorescence Activated Cell sorting (Flow Sorting) Expi293F cells (also referred to as “ExpiHEK cells”, ThermoFischer) are HEK293 cells that are adapted to grow in suspension. Expi293F cells were transfected with Gibco Expifectamine transfection kit following manufacturer protocols. Briefly, cells at a density of ~3 x 106 / mL were transfected with NaV channel constructs (tagged with -P2A-eGFP) and either UbiquiNav-P2A-mCherry or mCherry. Transfection enhancing solution was added to cell suspensions 24 hours after plasmid addition as per manufacturer protocol. 48 hours after plasmid addition, cells were harvested for Fluorescence Activated Cell Sorting (FACS) on WOLF G2 Cell Sorter (Nanocellect). FACS was conducted with cells resuspended in ExCell media. A minimum of 500,000 cells were collected in each condition. For ND7 / 23 cells, transfection was achieved with the Lipofectamine 2000 reagent following manufacturer protocols. Cells were transfected with eGFP-2A-NaV1.8 and either UbiquiNav-2AmCherry or mCherry.48 hours later, cells were dissociated with the use of Accutase. FACS was conducted with cells resuspended in ExCell media containing 5 U / mL DNase I and 5 mM MgCl2. Sheath fluid was identical to sample fluid. A minimum of 500,000 cells45678892.179 ATTORNEY DOCKET NO. YU 8723 PCT were collected in each condition. After FACS, samples were manually inspected to ensure >95% of cells were doubly positive for green and red fluorescence. Two independent transfections, sorts, and automated patch runs were conducted for each channel construct. Automated Electrophysiology – Heterologous Systems Cells were resuspended in 3 mL of extracellular recording solution (see below) before placement in the 1 well Cell Transfer Plate. Sodium currents were measured in the whole-cell voltage-clamp configuration using a Qube-384 (Sophion A / S, Copenhagen, Denmark) automated patch-clamp (APC) system. Intracellular solution contained (in mM): 120 CsF, 10 NaCl, 2 MgCl2, 10 HEPES, adjusted to pH 7.2 with CsOH. The extracellular recording solution contained (in mM): 145 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 10 HEPES, adjusted to pH 7.4 with NaOH. Liquid junction potentials calculated to be ~7 mV were not adjusted for. Currents were low pass filtered at 5 kHz and recorded at 25-kHz sampling frequency. Series resistance compensation was applied at 100% and leak subtraction enabled. The Qube-384 temperature controller was used to maintain recording chamber temperature for all experiments at 22 ± 2°C at the recording chamber. Appropriate filters for cell membrane resistance (typically >500 MOhm), series resistance (<10 MOhm), and NaVcurrent magnitude (>500 pA at a test pulse from a resting HP of −120 mV) were routinely applied to exclude poor quality recordings. The experimenter responsible for conducting automated patch clamp experiments and analysis was blinded to the conditions of the transfection. A separate experimenter assigned labels to the data after unblinding. Automated Electrophysiology – Primary Neurons DRGs (at least 24 DRGs from each mouse) were harvested and immediately put in ice- cold complete saline solution (CSS) (in mM: 137 NaCl, 5.3 KCl, 1 MgCl2, 25 sorbitol, 3 CaCl2, and 10 HEPES, adjusted to pH 7.2 with NaOH). After all the DRGs were harvested, DRGs were transferred to 37°C enzyme solution - 0.5 U / mL Liberase TM (Roche) and 0.6 mM EDTA in CSS for a 20-minutes incubation at 37°C, followed by a 15-minutes incubation at 37°C in another enzyme solution - 0.5 U / mL Liberase TL (Roche), 0.6 mM EDTA, and 30 U / mL papain (Worthington Biochemical) in CSS. DRGs were then centrifuged and triturated in 0.5 mL of 1.5 mg / mL BSA (low endotoxin) and 1.5 mg / mL trypsin inhibitor (Sigma) in DRG media [DMEM / F12 (Invitrogen) with 100 U / ml penicillin, 0.1 mg / ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), and 10% fetal bovine serum (Hyclone)]. After trituration, un- dissociated pieces were removed by filtering through a 70-μm mesh (Becton Dickinson). To remove small supporting cells and small pieces of dissociated axons and myelin, density gradients with 15% BSA were applied twice. Cells were pelleted and re-suspended with 1 ml45678892.180 ATTORNEY DOCKET NO. YU 8723 PCT DRG media, layered on top of 15% BSA solution and centrifuged at 250 g for 10 min at 4°C; pelleted cells were then re-suspended with DRG media and went through a second round of 15% BSA purification. The cell pellet was re-suspended with 1 ml of DMEM / F12 (4°C) to get single- cell suspension. Five µl of cell suspension was counter stained with five µl of trypan blue to check neuronal number, viability, and purity. Single-cell suspension [225±75 K (mean±SD) live neurons total] was taken for FACS and cells were resuspended in ExCell media containing 5 U / mL DNase I and 5 mM MgCl2. Sheath fluid was identical to sample fluid. A minimum of 50,000 cells were collected in each condition. FACS was conducted at 4° C to ensure membrane integrity. After FACS, samples were manually inspected to ensure >95% of cells were doubly positive for green fluorescence. Post-FACS neurons were diluted to 2.8 ml in DMEM / F12 (4°C) before delivering to the 384 well chip on the Qube-384 instrument (Sophion A / S, Copenhagen, Denmark). An aliquot of the cell suspension was plated on a 35-mm Tissue Culture-treated culture dish and imaged using a Nikon microscope (Eclipse TE2000-U Inverted Microscope). Sodium currents were measured in the whole-cell configuration using a Qube-384 automated voltage-clamp system (VH = -80 mV). Intracellular solution contained (in mM): 120 CsF, 10 NaCl, 2 MgCl2, 10 HEPES, adjusted to pH7.2 with CsOH. The extracellular recording solution contained (in mM): 145 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 10 HEPES, adjusted to pH7.4 with NaOH.1 µm TTX was perfused to isolate NaV1.8 channels. Liquid junction potentials calculated to be ~7 mV were not adjusted for. Currents were low pass filtered at 5 kHz and recorded at 25 kHz sampling frequency. Series resistance compensation was applied at 100% and leak subtraction enabled. The Qube-384 temperature controller was used to maintain recording for all experiments at 22 ± 2 ˚C at the recording chamber. Appropriate filters for series resistance (<10 MOhm) and NaV current magnitude (more than baseline in the inward direction, 0 nA) were routinely applied to exclude poor quality recordings. Data analysis was performed using Analyzer (Sophion) and Prism (GraphPad Software Inc., La Jolla, CA, USA) software. To determine the voltage-dependence of activation, we measured the peak current amplitude at test pulse potentials ranging from −120 mV to +30 mV in increments of +5 mV for 500 ms. Current-clamp electrophysiology Recordings from rodent DRG neurons were taken 24-48 hours post-transfection. Extracellular bath solution contained (in mM): 140 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 dextrose, and 10 HEPES. Osmolarity was brought to approximately 320 mOsm with sucrose and the pH was titrated to 7.3 with NaOH. Intracellular pipette solution contained (in mM): 140 KCl, 3 Mg-45678892.181 ATTORNEY DOCKET NO. YU 8723 PCT ATP, 0.5 EGTA, 5 HEPES, and 20 dextrose. Osmolarity was similarly adjusted to approximately 320 mOsm and the pH was adjusted to 7.3 using KOH. Current-clamp recordings were sampled at 50 KHz and filtered using two Bessel filters at 10 and 2.9 KHz. DRG neurons with an input resistance lower than 100 MΩ were excluded from analysis. Input resistance was determined by the slope of a linear fit to hyperpolarizing responses to current steps from −5 pA to −40 pA in −5 pA increments. Action potential repetitive firing was determined by summing the total number of action potentials that a neuron fired after a 1 second current injection. Current threshold was defined as the first current injection step that resulted in action potential firing without subsequent failure and was determined by a series of depolarizing current injections (200 ms) that increased incrementally by 5 pA. For the calculation of threshold, action potentials were defined as rapid increases in membrane potential to >40 mV with a total amplitude >100 mV. However, as neurons often attenuate firing with repetitive action potential spiking, when examining repetitive firing, action potentials were counted if the membrane potential rapidly crossed 0 mV, regardless of overshoot or total amplitude. Action potential repetitive firing was determined by summing the total number of action potentials that a neuron fired after a 1 second current injection. Dynamic clamp recordings of Nav1.8-null DRG neurons Current clamp procedures were the same as described above. DRG neurons from NaV1.8- null mice were harvested and transfected as described. A Hodgkin & Huxley kinetic model of NaV1.8 was derived from published literature5. DRG neurons were dynamically clamped using the Cybercyte DC1 dynamic clamp system (Cytocybernetics, Buffalo NY). The amount of NaV1.8 current injected into each neuron was specific to the capacitance of each cell, as described in previous literature6. In brief, the NaV1.8 channel model was based on Hodgkin-Huxley equations: ^^ = ^^− − ^^^ where m and h represent channel gates, and α and β are forward and backward rate constants, respectively. These rate constants were voltage-dependent and defined by the following equations:45678892.182 ATTORNEY DOCKET NO. YU 8723 PCT 7.35 ^^= 7.35 − ^^^.() (1 + ' ^*.+ 5.97 ( .* / ^^.(0 *.-. ^"= − Imaging As described previously7–11, microfluidic chambers (MFCs) were bound to glass- bottomed dishes according to manufacturer's instructions. Dissected DRGs were transfected as described previously10,12and plated into the somatic chamber of prepared MFCs containing DRG media with growth factors. Media in the axonal chambers was supplemented with 2x growth factors. After 24 hours, medium was changed to serum free-medium in both chambers. After 5-7 days in vitro, DRG neurons were taken for imaging experiments. For experiments involving TNF-α or Paclitaxel, neurons were incubated in either 20 ng / mL TNF-α, 50 nM PTX, or DMSO control for 24 hours prior to imaging. Neurons were washed with DRG-Neuronal Imaging Saline (NIS) for these experiments. DRG-NIS contained (in mM): 136 NaCl, 3 KCl, 1 MgSO4, 2.5 CaCl2, 0.15 NaH2PO4, 0.1 Ascorbic Acid, 20 HEPES, 8 Dextrose (pH 7.4 with NaOH, adjusted to 320 mOsm / l). ((May use media for all imaging experiments, modify in this case)). Surface Expression Halo-NaV1.8 channels at the somatic and axonal surface were labeled by incubation with cell-impermeable JF549i-HaloTag ligand. Both somatic and axonal compartments were thoroughly washed with DRG-NIS, fixed with 4% paraformaldehyde, and then taken for confocal fluorescence imaging. The fluorescence signal intensity of surface-labeled Halo-NaV1.8 from compressed confocal z-stacks of the distal 50 µM of axons was measured. Trafficking – OPAL imaging Trafficking assays in these studies were variations on the Optical Pulse-chase Axonal Long-distance (OPAL) imaging method described previously7,8,11.100 nm cell-permeable JFX650-Halo ligand was applied to the somatic chamber of MFCs containing DRG neurons transfected with Halo-NaV1.8 and UbiquiNaV constructs. After 15 minutes of incubation, the45678892.183 ATTORNEY DOCKET NO. YU 8723 PCT chambers were thoroughly washed with NIS, and MFCs were placed in a 37° C stage-top incubator for imaging. Halo-NaV1.8 positive neurons were identified by red fluorescence (from the mRuby2 reporter) and UbiquiNaV positive neurons were identified by green fluorescence (eGFP reporter). Multiple fields of view in the axonal chamber were selected, and anterogradely trafficking vesicles were imaged in the far-red channel continuously for 2 minutes. Resulting movies were opened in ImageJ and the KymographClear toolset was used to create kymographs of the selected axons. Axons containing anterogradely-moving vesicles were traced manually using a 50 µm segmented line, and the signal under that line was converted into a two-dimensional image that could be further analyzed. KymoButler is a machine learning algorithm that traces vesicle tracks from kymographs. Vesicle flux was determined by counting the number of vesicles which crossed the midline of the kymograph. Vesicle intensity was determined as the average of fluorescence values of pixels along the vesicle track, minus the background signal of the kymograph (defined as the modal value for that kymograph). The fluorescence intensities of multiple vesicles within axons were averaged.CoTrafficking – OPAL imaging Cotrafficking assays in these studies were variations on the Optical Pulse-chase Axonal Long-distance (OPAL) imaging method described previously7,9,10.100 nm cell-permeable JFX650-Halo ligand and 100 nm cell-permeable JFX554cp-SNAP ligand were applied to the somatic chamber of MFCs containing DRG neurons from NaV1.8-null mice transfected with Halo-NaV1.8 and SNAP-UbiquiNaV-C942S constructs. Anterogradely moving vesicles containing SNAP-UbiquiNaV-C942S were identified, and axons within the axonal chamber were then imaged in red and far-red by rapid laser and color filter switching. Kymographs were generated as above, and kymographs from each fluorescent channel were analyzed to score vesicles as positive for one or both proteins. The background was measured for each color and kymograph and subtracted from the vesicle measurements. A cutoff of 100 A.U. was used to categorize vesicles as positive or negative for each protein. Molecular Biology Plasmids Sclt1: Rat Sclt1 (also known as CAP-1A) was previously reported13. Briefly, the complete coding sequence (688 amino acids) was subcloned in-frame into the NheI / BamHI sites of pEGFP-N1 vector (Clontech) to produce plasmid pCAP-1A-GFP. Full-length Rat SCLT1 cDNA generated by double digestion of pCAP-1A-GFP with NheI / AgeI was subcloned into the XbaI / AgeI sites of pcDNA3.1B (Invitrogen), and the native translation termination codon was45678892.184 ATTORNEY DOCKET NO. YU 8723 PCT restored by site directed mutagenesis using the QuickChange system (Stratagene, La Jolla, CA) to produce the vector pcDNA3-CAP-1A (Referred to as rSCLT1 hereinafter). UbiquiNav: A bifunctional molecule, UbiquiNav was generated, using the SCLT1-MTD as the binding domain and the HECT domain as the catalytic ubiquitinating domain. Using molecular biology techniques a construct that encodes the SCLT1-MTD was assembled (a.a. 249-441) fused in frame with the HECT domain from human NEDD4-2 (a.a.594-974) and the P2A-GFP tag. The virus P2A sequence encodes a 33 amino acid linker that permits the production of two independent proteins from the same mRNA by causing the cleavage of the linker joining the upstream protein (SCLT1-MTD-HECT) and the downstream protein GFP54,55. UbiquiNav produced in multiple steps: First, the sequence that encodes the MTD module in rat SCLT1 [aa 249-441] was subcloned into the pEGFP-N1 vector using standard cloning methods24. The MTD-P2A-eGFP construct was created by inserting 135bp P2A into the MTD-eGFP construct by mega mutagenesis using the following primers and standard PCR amplification protocols: Second, The 1200bp fragment that encodes the HECT domain in NEDD4L (NEDD4.2) was amplified from the plasmid pCMV6-XL4-Nedd4.2 using the following primers and standard PCR protocols: The amplicon was then inserted by mega mutagenesis into MTD-P2A-eGFP to create the MTD-HECT-P2A-eGFP construct (UbiquiNav). The eGFP fluorescent protein in MTD-P2A-eGFP was exchanged with mRuby using mega mutagenesis to produce MTD- P2A-mRuby. The HECT sequence was inserted immediately downstream of the P2A using mega mutagenesis to generate the MTD-P2A-HECT-mRuby2. The eGFP fluorescent protein in the MTD-HECT-P2A-eGFP construct was exchanged with mCherry using mega mutagenesis to produce MTD-Hect-P2A-mCherry UbiquiNav. The identity of the construct was verified by sequencing of the insert. The nucleic acid sequence for MTD-HECT-P2A-mCherry is represented as SEQ ID NO:23 below. The MTD domain determines specificity to interaction with Nav1.8 channels and is represented by the uppercase bolded nucleotides in SEQ ID NO:23. HECT (Homologous to the E6-AP Carboxyl Terminus) is the catalytic domain of NEDD4-2 ubiquitin ligase and is represented by the uppercase underlined nucleotides in SEQ ID NO:23. P2A encodes is the amino acid linker that permits independent production of the upstream and downstream proteins and is represented by the lowercase italicized nucleotides in SEQ ID NO:23. mCherry is the reporter protein to identify transfected cells; and is represented by the uppercase italicized and bolded nucleotides45678892.185 ATTORNEY DOCKET NO. YU 8723 PCT in SEQ ID NO:23. In some forms, mCherry is replaced by eGFP. An exemplary linker is represented by the nucleotide sequence in lowercase letters (not italicized). The linker is inserted into the construct to provide flexibility between the two functional proteins. ATGACTGAAGGTCTTCAAGAGCAGATGCTGAAGAAGGAGGAAGATATTA TGTCTGCACAAGGAAAAGAGGAAGCATCAGATAGACGTGTGCAGCAGTTACAG TCTAGTATAAAACAGTTAGAGTCAAGATTGTGTATAGCAATTCAAGAAGCCAAT GTACTAAAAACAGGGAAAACACAATTGGAAAAGCAGATCAAAGAGCTACAAGC AAAGTGCAGTGAATCAGAAAATGAGAAATATGAGGCTATTTCAAGAGCCAGAG ATAGCATGCAGCTGTTAGAAGAAGCTAACATTAAACAGAATCAGATTCTACTTG AAGAGAAGCAAAAAGAGGTTGAAAGAGAAAAAATGAAGAAAACAATTTCTCAT CTTATACAAGATGCCGCCATCAAAGCAAGGAAAGAAGTTGAAAGCACAAAAAA ACAGTATGAAGTACTAATTTTGCAGTTAAAGGAAGAACTCTCAGCCCTTCAGAT GGACTGTGATGAAAAGCAAGGTCAAATTGATCGAGCCATTAGAGGGAAAAGAG CTGTGGAAGAAGAACTCGAGAAGATTTATCGTGAAGGCAAACAGGATGAAGGG ggatccaccggtTCCAGAGAATTTAAGCAGAAATATGACTACTTCAGGAAGAAATTAAAG AAACCTGCTGATATCCCCAATAGGTTTGAAATGAAACTTCACAGAAATAACATATTT GAAGAGTCCTATCGGAGAATTATGTCCGTGAAAAGACCAGATGTCCTAAAAGCTAG ACTGTGGATTGAGTTTGAATCAGAGAAAGGTCTTGACTATGGGGGTGTGGCCAGAG AATGGTTCTTCTTACTGTCCAAAGAGATGTTCAACCCCTACTACGGCCTCTTTGAGT ACTCTGCCACGGACAACTACACCCTTCAGATCAACCCTAATTCAGGCCTCTGTAATG AGGATCATTTGTCCTACTTCACTTTTATTGGAAGAGTTGCTGGTCTGGCCGTATTTCA TGGGAAGCTCTTAGATGGTTTCTTCATTAGACCATTTTACAAGATGATGTTGGGAAA GCAGATAACCCTGAATGACATGGAATCTGTGGATAGTGAATATTACAACTCTTTGAA ATGGATCCTGGAGAATGACCCTACTGAGCTGGACCTCATGTTCTGCATAGACGAAG AAAACTTTGGACAGACATATCAAGTGGATTTGAAGCCCAATGGGTCAGAAATAATG GTCACAAATGAAAACAAAAGGGAATATATCGACTTAGTCATCCAGTGGAGATTTGT GAACAGGGTCCAGAAGCAGATGAACGCCTTCTTGGAGGGATTCACAGAACTACTTC CTATTGATTTGATTAAAATTTTTGATGAAAATGAGCTGGAGTTGCTCATGTGCGGCC TCGGTGATGTGGATGTGAATGACTGGAGACAGCATTCTATTTACAAGAACGGCTACT GCCCAAACCACCCCGTCATTCAGTGGTTCTGGAAGGCTGTGCTACTCATGGACGCCG AAAAGCGTATCCGGTTACTGCAGTTTGTCACAGGGACATCGCGAGTACCTATGAAT GGATTTGCCGAACTTTATGGTTCCAATGGTCCTCAGCTGTTTACAATAGAGCAATGG GGCAGTCCTGAGAAACTGCCCAGAGCTCACACATGCTTTAATCGCCTTGACTTACCT CCATATGAAACCTTTGAAGATTTACGAGAGAAACTTCTCATGGCCGTGGAAAATGCT45678892.186 ATTORNEY DOCKET NO. YU 8723 PCT CAAGGATTTGAAGGGGTGggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaac cctggacctATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATG CGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGC GAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAA GGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCC AAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCG AGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGA CCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCA CCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCT CCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGC TGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCA AGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTC CCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTC CACCGGCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO:23). The amino acid sequence for MTD-HECT-P2A-mCherry is represented as SEQ ID NO:24 below. The MTD sequence determines specificity to interaction with Nav1.8 channels and is represented by the uppercase bolded amino acids in SEQ ID NO:23. HECT is the catalytic domain of NEDD4 ubiquitin ligase and is represented by the uppercase italicized amino acids in SEQ ID NO:23. P2A is the amino acid linker that permits independent production of the upstream and downstream proteins and is represented by the lowercase italicized amino acids in SEQ ID NO:1. mCherry is the reporter protein to identify transfected cells; and is represented by the uppercase italicized and bolded amino acids in SEQ ID NO:23. mCherry can be replaced by eGFP. An exemplary linker is represented by the amino acid sequence in lowercase underlined letters. The linker is inserted into the construct to provide flexibility between the two functional proteins. MTEGLQEQMLKKEEDIMSAQGKEEASDRRVQQLQSSIKQLESRLCIAIQEA NVLKTGKTQLEKQIKELQAKCSESENEKYEAISRARDSMQLLEEANIKQNQILLEE KQKEVEREKMKKTISHLIQDAAIKARKEVESTKKQYEVLILQLKEELSALQMDCD EKQGQIDRAIRGKRAVEEELEKIYREGKQDEGgstgSREFKQKYDYFRKKLKKPADIPNR FEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKGLDYGGVAREWFFLLSKEMFNPYY GLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAVFHGKLLDGFFIRPFYKMMLGK QITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQTYQVDLKPNGSEIMVTNENK REYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELELLMCGLGDVDVNDWRQH SIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQLFTIE45678892.187 ATTORNEY DOCKET NO. YU 8723 PCT QWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVgsgatnfsllkqagdveenpgp MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQ YERAEGRHSTGGMDELYK (SEQ ID NO:24). Inducible UbiquiNav: The Mandipropamid dimerization system is described in Reference 29. The PYR1 module was synthesized according to the sequence provided in Ref.29 and cloned into pUC57 (GenScript). Then 648bp PYR1 was amplified from pUC57-PYR1 & inserted by mega mutagenesis MTD-P2A-mRuby2 to create MTD-PYR1-P2A-mRuby2. The SV-ABAactDA construct was obtained from Addgene (Plasmid #38247) and used as a template to amplify a 957bp fragment encoding ABI1 using standard PCR protocols. This ABI1 fragment was exchanged for the mRuby in MTD-P2A-HECT-mRuby2 by mega mutagenesis to create MTD-P2A-HECT-ABI1. The 1435bp fragment encoding PYR1-P2A-mRuby2 was amplified from MTD-PYR1- P2A-mRuby using standard PCR protocols, and inserted by mega mutagenesis into MTD-P2A- HECT-ABI1 to create MTD-PYR1-P2A-mRuby2-P2A-HECT-ABI1 (iUbiquiNav). UbiquiNav in AAV shuttle vector: pFBAAV-MTD-HECT-P2AeGFP was created by cloning a 2534bp EcoRI-NotI fragment from MTD-HECT-P2A-eGFP into 5761bp EcoRI-NotI digested pFBAAVCMVmcswtIRESeGFPBghpA (from University of Iowa Viral Vector Core #G0692). This shuttle vector will be sent to UPenn virus core to generate recombinant AAV9 capsids that produce UbiquiNav. Human Nav1.8: The codon-optimized human Nav1.8 construct (pcDNA5-SCN10A) was purchased from Genionics, and previously reported (Faber et al.(2012) Proc Natl Acad Sci USA 109:19444–19449)9. The final construct topology is in order from the N-terminus:1-30 a.a. β 4 signal peptide, 3 myc tag (EQKLISEEDL; SEQ ID NO:19), Halo-tag enzyme (297 a.a.) (Promega), 3 HA tag (YPYDVPDYA; (SEQ ID NO:31)), 30 a.a. extracellular stalk (β 4132- 162), 21 a.a. transmembrane segment (β 4163-183), 7 a.a. linker (SGLRSAT (SEQ ID NO:32)), hNaV1.8. Human Nav1.8 / Nav1.7 chimera: The eGFP-2A-h1.8RD1-4_h1.7C was created by replacing the 693bp C-term of h1.8R in the codon-optimized eGFP-2A-hNaV1.8R with the 733bp C-term of h1.7 using mega mutagenesis and standard PCR protocols. AAV Production: AAV9 carrying CMV-eGFP-2A-UbiquiNav was produced by the Penn Vector Core. The final titer of the virus was 7.13e13 GC.45678892.188 ATTORNEY DOCKET NO. YU 8723 PCT Statistics Unless noted otherwise, the following two-stage statistical procedure for all group comparisons was performed. First, testing for normality using the Shapiro-Wilks test and the Kolmogorov-Smirnov were performed. If the underlying data distribution was normal, parametric statistical testing using Student’s t-test was performed. If the distribution of data was non-normal, groups were compared using non-parametric testing using the Mann-Whitney U- test14. For repeated measures data, groups were compared using mixed effects modeling. A level of significance α=0.05 was used with p-values less than 0.05 considered to be statistically significant. When multiple comparisons were made, the False Discovery Rate was quantified, and p-values were adjusted accordingly to correct for multiple comparisons. All values are reported as means ± standard error of means (SEM). Results Development of a bifunctional protein that binds NaV1.8 and tags it for degradation A heterobifunctional protein that enables targeted degradation of a membrane protein requires a selective binding module and a ubiquitinating module (Figure 1A, Schematic). It was previously shown that the protein Sclt1 binds the C-terminus of NaV1.8 selectively over all other NaV isoforms. Additionally, a module of the Sclt1 protein that shares homology to the Myosin Tail Domain (MTD) is necessary and sufficient for this binding13. Overexpressing Sclt1 on its own reduces endogenous NaV1.8 currents in rat pup DRG neurons (Sclt1: 123.3 ± 22.5; GFP: 288.8 ± 37.5 pA / pF, p<0.01; Figures 1B-1D) and overexpressing MTD alone does not result in a similar decrease in the Nav1.8 current MTD: 267.7 ± 63.8; GFP: 191.4 ± 27.47 pA / pF, p>0.05; Figures 1E-1G ). To evaluate the effect of Sclt1 on the human NaV1.8 channel isoform, DRG neurons from NaV1.8-null mice were transfected with plasmids encoding human NaV1.8 in addition to either Sclt1 or GFP-control. Overexpression of rodent Sclt1 reduced human NaV1.8 current amplitude and density (Figure 1H). NEDD4 is a ubiquitin ligase shown to tag NaV1.8 and other sodium channel isoforms for degradation15, 16. Overexpression of NEDD4 in neurons results in a non-selective reduction of multiple types of ion channels and other membrane proteins17,18. It has been shown that attachment of the NEDD4L (also known as NEDD4-2) catalytic HECT domain to a nanobody specific for neuronal Ca2+-channels enables the ubiquitination of these channel targets19,20. The HECT domain of NEDD4L was attached to the MTD to generate UbiquiNav (Figure 1I, top panel); the data showed that steady state UbiquiNav levels could be detected by western blot in transfected HEK293 cells (Figure.1I, bottom panel). The UbiquiNaV construct contains an eGFP reporter linked in frame by a P2A linker to enable identification of positively transfected45678892.189 ATTORNEY DOCKET NO. YU 8723 PCT cells. This indicates that UbiquiNav does not degrade itself by autoubiquitination. Together, these findings indicate that UbiquiNav is a heterobifunctional peptide that binds to NaV1.8 channels and results in their ubiquitination. UbiquiNav reduces NaV1.8 current density, surface expression, and delivery of NaV1.8 channels in distal axons in rat pup DRG neurons To evaluate the effects of UbiquiNav on NaV1.8 function in sensory neurons, DRG neurons isolated from 2-4 d old rat pups (P2-4 rat pup DRG neurons) were transfected with plasmids encoding UbiquiNav, eGFP (to control for the effects of transfection), or a catalytically inactivated UbiquiNav (C942S), and the effects on endogenous NaV1.8 currents were recorded by whole cell voltage-clamp recordings (Figures 2A and 2B). DRG Neurons expressing UbiquiNav had significantly reduced NaV1.8 current amplitude and current density compared to neurons transfected with an eGFP control (172.5 ± 37.3 vs.368.4 ± 62.5 pA / pF; p<0.05; Figure 2C). Catalytic inactivation of UbiquiNav ablated these effects on NaV1.8 current density (434.6 ± 103.3 pA / pF, p<0.05 vs. UbiquiNaV, p>0.05 vs. eGFP-control; Figure 2C).. Electrophysiology is a powerful approach for determining the functional consequences of channel ubiquitination. However, the technique evaluates channel function in the somas of neurons. Sensory afferents in vivo are pseudounipolar neurons, each with a long axonal process that extends to the periphery. This distal axonal terminal is the site for initiation of action potentials21. Therefore, a strategy that aims to reduce sodium channel surface expression must accomplish this goal at the distal axon. Using a full length NaV1.8 construct tagged with the HaloTag enzyme (Halo-NaV1.83,9, Figure 2D), the impact of UbiquiNaV expression on elements of the NaV1.8 life cycle in sensory neurons was evaluated. By plating DRG Neurons in microfluidic chambers (MFCs), axons of DRG neurons from their cell bodies were fluidically isolated (Figure 2E). This approach enabled the evaluation of surface levels of Halo-NaV1.8 in both somas and axons7,10. It was investigated whether Halo- NaV1.8 levels at the DRG soma were affected by the presence of UbiquiNaV. A cell impermeable Halo ligand was applied to the somatic chambers of DRG neurons expressing Halo-NaV1.8 in glass bottom dishes and then fixed them with PFA (data not shown). Compared to neurons transfected with eGFP control, neurons transfected with UbiquiNaV exhibited significantly lower levels of Halo-NaV1.8 at the somatic surface (461.3 ± 62.0 vs.1628.0 ± 268.1 A.U., p<0.01; Figure 2F). This recapitulates voltage-clamp data which comes from somatic recordings. Using the same labeling approach as above but this time in the axonal chamber of MFCs containing DRG neurons, it was found that neurons expressing UbiquiNaV had significantly less45678892.190 ATTORNEY DOCKET NO. YU 8723 PCT Halo-NaV1.8 channels at distal axonal distal membranes compared to control (66.6 ± 5.6 vs. 131.5 ± 15.0 A.U., p<0.0001; Figure 2G). This reduction in distal axonal expression of Halo-NaV1.8 could be a manifestation of the action of UbiquiNaV at multiple stages of the channel lifecycle. It was assessed whether changes in channel delivery underlie the changes in distal surface expression of NaV1.8 induced by UbiquiNaV expression. How the trafficking of Halo-NaV1.8 channels was impacted by the expression of UbiquiNaV was investigated using the OPAL imaging technique. By adding cell- ¬permeable fluorophore conjugated Halo ligands to the somatic chamber of MFCs containing neurons transfected with Halo-NaV1.8, trafficking vesicles carrying labeled Halo-NaV1.8 channels in axons at a very high signal / noise ratio could be visualized anterogradely (Figure 2H). It was observed that the flux of vesicles carrying Halo-NaV1.8 was significantly reduced in the presence of UbiquiNaV (4.2 ± 0.9 vs.10.4 ± 1.0 vesicles / axon / min; data not shown, Figure 2I). Further studies leveraged a chemically induced dimerization strategy53. Briefly, each module of UbiquiNav (the MTD and the HECT) was attached to a separate peptide binding domain of the anti-fungal small molecule Mandipropamid. Each of these domains bind orthogonal areas of the Mandipropamid small molecule. Thus, in the absence of Mandipropamid, UbiquiNav would not be active since the MTD and the HECT domain would not associate with one another. The two halves are cloned in cis separated by a P2A-eGFP-P2A linker cassette which permits the translation of the three proteins independently (Figure 2J). When 1 mM Mandipropamid is added however, the MTD and HECT domains combine, yielding a functionally active UbiquiNav molecule (Figure 2J). This association is rapid (minutes), and thus enables the interrogation of the temporal dynamics of UbiquiNav activity. This construct was named inducible-UbiquiNav (iUbiquiNav). Following baseline recordings, DRG neurons expressing iUbiquiNav or GFP-control were perfused with 1 uM Mandipropamid. Every 10 seconds, a voltage step to the peak of activation (matched to the particular cell) was applied. Currents are normalized to the peak inward current at sweep 1. Mandipropamid was applied at the 5th sweep. Before perfusion of neurons with Mandipropamid, there was no significant difference in NaV1.8 current density or amplitude in rat pup DRG neurons transfected with iUbiquiNav vs. control (data not shown). After baseline 1 mM Mandipropamid was perfused, and current responses to 100 ms voltage- pulses were recorded to the peak of activation for each neuron. Current was recorded from 1 sweep every 10 seconds. Within 1 minute, decreases in NaV1.8 current amplitude were detected. Within ~30 minutes, NaV1.8 current amplitude was reduced to around 50% of baseline, and45678892.191 ATTORNEY DOCKET NO. YU 8723 PCT within 1 hour, it was reduced to around 10%. By contrast, DRG neurons transfected with GFP control retained ~75% of their NaV1.8 current amplitude 1-hour post-Mandipropamid perfusion (Figure 2K). To determine if UbiquiNaV travels in vesicles to distal axons, we generated a SNAP- tagged UbiquiNaV construct (Figure 6A). The SNAPTag functions similarly to HaloTag, but binds different cognate ligands which can be co-incubated with Halo ligands. We then transfected NaV1.8-null mouse DRG neurons with Halo-NaV1.8 and SNAP-UbiquiNaV-C942S and evaluated co-trafficking of these proteins with OPAL imaging (Figure 6B, data not shown). The inactivation of the UbiquiNaV construct was necessary to allow anterograde trafficking of Halo-NaV1.8 to travel into the distal axonal compartment since the expression of the active UbiquiNav significantly reduces the levels of the Nav1.8 in both soma and axons. Data shows that >90% of vesicles positive for SNAP-UbiquiNaV-C942S are positive for Halo- NaV1.8 (Figure 6C). Additionally, when there was no Nav1.8 channels either endogenous channels or Halo-NaV1.8 co-transfected with the SNAP-UbiquiNaV-C942S construct, there were no anterogradely trafficking vesicles carrying SNAP-UbiquiNaV-C942S in the axonal chamber (data not shown), whereas robust SNAP-UbiquiNaV-C942S could be detected with Halo- NaV1.8 co-transfection (data not shown). This data shows that axonal transport of UbiquiNav is dependent on the presence of Nav1.8 channels. Expression of UbiquiNav normalizes delivery and distribution of NaV1.8 channels and neuronal excitability in hyperexcitable rat pup DRG neurons Electrophysiological recordings show that UbiquiNav clearly reduces NaV1.8 channel surface expression and current density. However, its viability as a potential analgesic depends on its ability to normalize nociceptor hyperexcitability. To evaluate this, current-clamp electrophysiology was used to investigate the firing properties of rat pup DRG neurons transfected with UbiquiNav (Figure 3A). At baseline, rat pup DRG neurons do not fire repetitive trains of action potentials5,11. However, treatment of cultures with TNF-α rapidly sensitizes neurons and mimics an inflammatory pain state11,22–24. After incubation with 40 ng / mL TNF-α for 24 hours, sensory neurons become hyperexcitable and fire multiple action potentials readily (4.2 ± 1.1 APs in response to 200 pA; Figure 3B).. In the presence of TNF-α, sensory neurons are hyperexcitable and fire multiple action potentials readily. In neurons expressing UbiquiNav, application of TNF-α did not evoke hyperexcitability and firing frequency in response to gradually increasing current injection was similar to control neurons treated with DMSO vehicle (0.7 ± 0.1 vs.1.2 ± 0.4 APs in response to 200 pA; p>0.05, p<0.01 vs. TNF-α) (Figure 3B). TNF-α decreases the45678892.192 ATTORNEY DOCKET NO. YU 8723 PCT threshold for AP firing, while neurons that express UbiquiNaV have similar AP thresholds to control neurons. TNF-α dramatically increases repetitive firing behaviors in response to increasing current injections. UbiquiNaV expression ablates this effect of TNF-α, and neuronal firing remains consistent with un-inflamed firing behavior when UbiquiNaV is on board. (Figure 3D). In inflammatory pain, cytokines like TNF-α drive pro-nociceptive ion channels to both somatic and axonal membranes7,8,11. To investigate if UbiquiNaV could normalize the delivery and distribution of NaV1.8 throughout neuronal compartments in the setting of inflammation, the surface expression of Halo-NaV1.8 channels was evaluated after incubation with TNF-α (data not shown). Incubation of neurons with TNF-α resulted in a significant accumulation of NaV1.8 channels at both somatic and axonal membranes. UbiquiNaV-expressing neurons exposed to TNF-α had greatly reduced expression of channels in both the cell body (838.9 ± 132.5 vs. 3787.0 ± 935.2 A.U., p<0.05; Figure 3E) and at distal axons (74.4 ± 8.3 vs.293.8 ± 76.9 A.U., p<0.01; Figure 3F). Microtubule inhibiting chemotherapeutic agents like Paclitaxel (PTX) frequently lead to the development of neuropathic pain25,26. One of the mechanisms by which neuropathic pain may develop in response to these agents is increased delivery of pro-nociceptive ion channels like NaV1.7 and NaV1.8 to distal axons3,27. To evaluate if UbiquiNaVexpression is protective against NaV1.8 accumulation in response to PTX treatment, the surface expression of Halo-NaV1.8 channels was evaluated following incubation with PTX in neurons expressing UbiquiNaVvs. eGFP-control (data not shown). UbiquiNaV expressing neurons demonstrated markedly lower surface expression of Halo-NaV1.8 at both somas (194.4 ± 39.44 vs.2187.0 ± 689.3 A.U., p<0.05; Figure 3G) and axons (200.3 ± 52.2 vs 545.0 ± 78.8 A.U., p<0.001; Figure 3H) in response to PTX treatment. Trafficking of NaV1.8 in these hyperexcitable states was also investigated and it was observed that TNF-α incubation increases vesicular delivery of NaV1.8 to distal axons (data not shown). UbiquiNaV expression significantly reduces the flux of vesicles carrying Halo-NaV1.8 to the distal axon following TNF-a incubation (6.9 ± 1.9 vs.17.0 ± 2.1 vesicles / axon / min, p<0.01; Figure 3I). Similar results in neurons incubated with Paclitaxel were also observed (data not shown) - vesicular trafficking of Halo-NaV1.8 to distal axons was significantly reduced in neurons expressing UbiquiNaV post-PTX treatment (18.4 ± 2.4 vs.6.7 ± 1.1 vesicles / axon / min, p<0.001; Figure 3J). UbiquiNav is selective for NaV1.845678892.193 ATTORNEY DOCKET NO. YU 8723 PCT NaV1.8 is an attractive target for pain relief because of its preferential expression in peripheral sensory neurons28-30. Isoform selectivity of a NaV1.8-targeting agent is of paramount importance because other NaV isoforms are expressed in critical electrogenic organs, such as the CNS and cardiac pacemaker cells31-33. In peripheral sensory neurons, NaV isoforms can be distinguished on the basis of their pharmacologic susceptibility to tetrodotoxin (TTX). NaV1.1, NaV1.6, and NaV1.7 are TTX-Sensitive (TTX-S) while NaV1.8 and NaV1.9 are TTX-Resistant (TTX-R). These channel isoforms have different biophysical properties, and NaV1.8 currents can be isolated by voltage-protocols that inactivate all TTX-S isoforms (Cummins, et al., J Neurosci 1997 Vol.17 Issue 10 Pages 3503-14. Accession Number: 9133375) (Figure 7A). The data showed that the expression of UbiquiNav did not have any effect on TTX-S current density (Figure 7B) in rat pup DRG neurons. (Figure 7C). An automated patch-clamp assay was designed to enable high-throughput, unbiased evaluation of the effect of UbiquiNav on human NaV isoforms (Figure 4A). Briefly, suspension HEK293(Expi293F) cells were transfected with plasmids encoding P2A-eGFP-tagged NaVchannel constructs and UbiquiNav-P2A-mCherry (or mCherry alone as a control).48 hours post transfection, FACS was used to isolate the population of cells that expressed fluorescence in both red and green channels, indicating successful co-transfection and expression. These cells were then assayed on the Sophion Qube 384 Automated Patch-Clamp (APC) robot. As a positive control, DRG-derived ND7 cells were transfected with plasmids encoding eGFP-P2A-hNaV1.8 and UbiquiNav-mCherry or mCherry control and the experiment proceeded with an identical FACS to APC procedure; ND7 / 23 cells were used because Nav1.8 channels do not produce a robust current in HEK293 cells. UbiquiNav did not affect the current-voltage relationships of NaV isoforms 1.1 (Figure 8A) 1.2 (Figure 8B), 1.3 (Figure 8C), NaV 1.4 (Figure 8D), NaV 1.5 (Figure 8E), 1.6 (Figure 8F) and Nav1.7 (Figure 8G), nor did it reduce the current density of NaV isoforms 1.1-1.7 (Figures 4B-4I). NaV channel isoform 1.9 does not express well in heterologous systems34. However, the reduction in NaV1.8 current density expected from UbiquiNaV was recapitulated in this assay (38.9 ± 6.5 vs.94.1 ± 18.0 pA / pF in UbiquiNaV vs. Control treated cells, respectively; p<0.05; Figure 8H, Figure 4I). Dynamic clamp is an electrophysiologic technique that enables injection of simulated ion channel conductance using a computer-cell interface. A computer model of NaV1.8 current was first constructed using empirically-determined parameters which simulates its actual recordings with high fidelity. DRG neurons from NaV1.8-null mice demonstrate reduced action potential amplitude and loss of repetitive firing ability. Subsequent experiments then dynamically clamped in physiologic levels of NaV1.8-simulated current back into these neurons18and found that this45678892.194 ATTORNEY DOCKET NO. YU 8723 PCT addition restores action potential amplitude and repetitive firing behavior (data not shown). Then, the action potential firing properties of DRG neurons taken from NaV1.8-null mice transfected with UbiquiNav were compared vs. control that had been dynamically clamped with NaV1.8 current. There was no difference in resting membrane potential, repetitive firing behavior, or action potential firing threshold (Figures 8I-8L) in DRG neurons transfected with UbiquiNav vs. control in the absence of physical NaV1.8 channels. Though these experiments provided strong evidence to support the notion that UbiquiNav’s effects are NaV1.8 dependent, dynamic clamp used a computer generated channel conductance. To tackle the problem of electrogenic specificity in another way, the C-terminus of NaV1.8 was swapped with the C-terminus of NaV1.7 (NaV1.8 / 1.7C), producing a chimeric channel (Figure 8M) with NaV1.8-like activation properties (Figure 8N) but was resistant to UbiquiNav activity since the binding site had been altered (Figure 8O). These data indicate that the C-terminus of Nav1.8 is necessary for the binding of UbiquiNav. Since the ability of UbiquiNaV to decrease NaV1.8 currents is critically dependent on the affinity of the MTD for the NaV1.8 C-terminus, it was hypothesized that the human SCLT1 MTD sequence would serve as a better warhead for a UbiquiNaVagainst the human Na¬V¬1.8 isoform. Indeed, the effect size of current depression of NaV1.8 channels elicited by UbiquiNaV with the human MTD (hUbiquiNaV) was greater than that from UbiquiNaVwith the rat MTD binding module (Figure 9). UbiquiNav regulates neuronal excitability in a NaV1.8 dependent manner The sensory neuronal electrogenisome is comprised of a wide variety of voltage-gated ion channels beyond just NaVchannels35–37. It is untenable to individually evaluate the effect of UbiquiNaV on all voltage-gated ion channels that are present in sensory neurons. However, the binding module of UbiquiNaVmay bind off-target to other components of the excitable machinery in the cell. To evaluate if this is the case, two parallel approaches were employed to determine if the reduction in neuronal excitability provided by UbiquiNaVis dependent on the presence of NaV1.8 channels and the binding of the agent (Figure 4J). Dynamic clamp is an electrophysiologic method that enables injection of simulated ion channel conductance using a computer-cell interface. A computer model of NaV1.8 that simulates its physiological activity with high fidelity was created (Figure 10A). DRG neurons from NaV1.8-null mice demonstrated reduced action potential amplitude and loss of repetitive firing ability (Figure 4J, top panel). Physiologic levels of NaV1.8-simulated current was then dynamically clamped back into these neurons and it was found that this addition restores action potential amplitude and repetitive firing behavior (Figures 10B-10C). Then, the action potential45678892.195 ATTORNEY DOCKET NO. YU 8723 PCT firing properties of DRG neurons taken from NaV1.8-null mice transfected with UbiquiNaV vs. eGFP-control that had been dynamically clamped with NaV1.8 current were compared. It was observed that there was no difference in resting membrane potential (-59.7 ± 1.8 vs. -57.6 ± 2.1 mV, p>0.05; Figure 4K), action potential firing threshold (79.4 ± 15.9 vs.90.0 ± 23.0 pA, p>0.05; Figure 4L), or repetitive firing behavior (p >0.05; Figure 4M) in DRG neurons transfected with UbiquiNaV vs. eGFP-control in the absence of physical NaV1.8 channels. Though these experiments provided strong evidence to support the notion that UbiquiNaV’s effects are NaV1.8 dependent, dynamic clamp is an artificial system. To tackle the problem of electrogenic specificity in another way, knowledge of the previously published binding site of the MTD – the C-terminus of NaV1.8 was leveraged. The C-terminus of NaV1.8 was swapped with the C-terminus of NaV1.7 (NaV1.8 / 1.7C), producing a chimeric channel (Figure 11A) that supported NaV1.8-like currents (Figure 11B) but was resistant to UbiquiNaVactivity since the binding site had been altered (Figure 11C). Transfecting DRG neurons from NaV1.8-null mice with this chimeric channel construct restored repetitive firing ability (Figure 4J, bottom panel). It was assessed if UbiquiNaV had any effects on excitability in NaV1.8-null mouse neurons expressing this UbiquiNaV-resistant NaV1.8 / 1.7C construct. Recapitulating results of the dynamic clamp experiments, it was found that UbiquiNaV did not affect resting membrane potential (-51.5 ± 2.2 vs. -53.4 ± 2.0 mV, p>0.05; Figure 4N), action potential firing threshold (127.5 ± 42.5 vs.97.7 ± 27.5 pA, p>0.05; Figure 4O), or repetitive firing behavior (p >0.05; Figure 4P) in NaV1.8-null mouse DRG neurons transfected with UbiquiNaVvs. eGFP- control along with NaV1.8 / 1.7C. AAV9-UbiquiNav can deliver UbiquiNaVto neurons in vivo and reduce NaV1.8 currents An AAV9 virus containing UbiquiNaV was generated to permit in vivo delivery of UbiquiNaVto sensory neurons. To test the function of the virus, whether AAV9-UbiquiNaVinfection would reduce NaV1.8 currents in rodent neurons in vitro was tested and verified (data not shown). The in vitro function of the virus in DRG neurons from a human donor was also tested and it was observed that AAV9-UbiquiNaV infection significantly reduces NaV1.8 currents in human DRG neurons (46.0 ± 5.8 vs.225.3 ± 83.3 pA / pF, p<0.001; Figure 5A). Whether the AAV9-UbiquiNaV could reduce NaV1.8 currents in vivo on a population level was assessed. Manual patch clamp techniques suffer from low throughput and operator bias. An automated patch clamp assay that would allow for the electrophysiological characterization of neurons from animals following intrathecal injection of an AAV was created (Figure 5B). C57BL / 6 mice were injected with 1012vg of AAV9-UbiquiNaV or AAV9-eGFP per mouse intrathecally.6 weeks after injection, lumbar DRG neurons from 4-5 mice were45678892.196 ATTORNEY DOCKET NO. YU 8723 PCT harvested and pooled. A series of BSA gradients38and FACS sorting were used to purify and isolate neurons that expressed the eGFP reporter protein in infected neurons, and then automated patch clamp experiments were conducted. DRG neurons that express UbiquiNaV demonstrate markedly lower NaV1.8 currents (14.7 ± 3.7 vs.3.9 ± 1.4 nA, p<0.01; Figure 5C) and current density (231.3 ± 73.2 vs.640.5 ± 48.2 pA / pF, p<0.001; Figure 5D) when compared to control. Discussion In this study, an agent, UbiquiNaV, that functions as a selective degrader of NaV1.8 channels in neurons of the dorsal root ganglia was described and tested. UbiquiNaVleverages the isoform selectivity of an intracellular binder of NaV1.8 and links it to the catalytic activity of a classical E3 ubiquitin ligase (Figures 1A-1I). This powerful combination enables the post- translational knockdown of NaV1.8 channels in DRG neurons. It was demonstrated that UbiquiNaVreduces NaV1.8 surface expression and current density (Figures 2A-2K) and consequently normalizes nociceptor hyperexcitability (Figures 3A-3J). UbiquiNaV acts to decrease NaV1.8 surface expression at both the neuronal cell body as well as the distal axon. The main action of UbiquiNaV appears to take place in the soma, as vesicular delivery of NaV1.8 channels to the distal axon is greatly dampened in the presence of UbiquiNaV. Through this activity, UbiquiNaV can normalize distribution and delivery of NaV1.8 channels throughout the complex sensory neuron, an important consideration for in vivo analgesia. Importantly, it was demonstrated that UbiquiNaV is selective for NaV1.8 over all mammalian NaV channels, and show using two parallel approaches that UbiquiNaVdoes not have off-target effects on any component of the neuronal electrogenisome (Figures 4A-4P). Additionally, UbiquiNaV proteins fail to travel to distal axons in the absence of NaV1.8, further indicating associative selectivity (Figures 6A-6C, data not shown). Small molecule blockers have reached endpoints in clinical trials for acute pain39. However, the delivery of these molecules (need to cross the blood-nerve barrier, continuous oral redosing, etc.) has been challenging, and this may contribute to relatively small clinical effect sizes of these agents40,41. In contrast, a gene therapy approach, though administered in a more invasive manner, has the potential to be a more effective and durable treatment for chronic pain. Intrathecal delivery is one appropriate delivery modality for this targeted degradation approach. NaV1.8 is primarily expressed in primary sensory afferents, whose cell bodies reside in the dorsal root ganglia. Compared to systemic delivery, intrathecal delivery has advantages in both transduction efficiency42and reduction of peripheral biodistribution43. The dose needed in an intrathecal injection is much lower than a peripherally administered dose, and the relative lack of immune cells in the intrathecal space may contribute to a potential lower immunogenicity of45678892.197 ATTORNEY DOCKET NO. YU 8723 PCT AAV injection. Further, the development of anti-AAV antibodies after AAV delivery44may be less problematic because viral redosing may not be necessary given the long-term expression of AAV-vector in non-dividing cells of the PNS45. However, caution must be taken with AAV vectors as studies in NHPs have revealed DRG toxicity in peripherally administered AAVs46and liver injury in both systemic and intrathecal delivery47.Future approaches may seek to move away from the AAV delivery modality, and instead utilize non-immunogenic genetic delivery methods (e.g., liposomal delivery). The results of the present study demonstrate selective reduction in NaV1.8 distribution with concomitant effects on nociceptor hyperexcitability. These results provide strong support for the development of this approach as an analgesic therapy. However, several pertinent limitations exist. For one, though we investigated pain modulation in rodent models, significant species differences exist in both NaVchannel expression as well as biophysics5. A different amount of NaV1.8 degradation, or an increased neuronal gene delivery rate may be necessary to elicit analgesia in the human pain setting. Additionally, UbiquiNaVexpression did not totally knock down NaV1.8 expression or currents. This may, however, actually be an advantage when compared to a CRISPR / Cas9 approach to gene silencing, since some NaV1.8 channels will remain, and nociceptive firing will still be possible. Indeed, the present results demonstrate that the main action of UbiquiNav is to normalize hyperexcitability, not ablate pain signaling entirely. Durability is also an important factor in the treatment of chronic pain; AAV-vectors can last for a very long time in non-dividing cells48and UbiquiNaVexpression should theoretically persist as long as the vector is active. The CMV promoter used in this study is silenced ~2 months post-infection49. Beyond its potential as a therapeutic agent, UbiquiNaV also has great utility as a research tool. Though there have been reports on the trafficking and delivery of ion channels7,9,50,51, there is relatively little knowledge of the latter stage of the channel life cycle; that is, what happens following channel internalization52. UbiquiNaV-triggered ubiquitination of NaV1.8 channels permits the study of what happens to channels that are marked for degradation, particularly when coupled with next-generation super resolution imaging modalities. Additionally, the design of UbiquiNaV serves as a prototype for the development of a new class of protein therapeutics that enable isoform-specific targeting of membrane proteins such as ion channels. Small molecule PROTAC design is challenging and isoform-specific binders of proteins such as the NaV channel family is often precluded by high sequence and structural similarity. On the other hand, peptides frequently interact with such proteins in an isoform-specific manner; the binding modules of these peptides that enable these specific interactions can be leveraged as warheads for protein45678892.198 ATTORNEY DOCKET NO. YU 8723 PCT ubiquitination or deubiquitination. The length of these peptides can be reduced to minimal fragments required for selective binding and the recruitment of proteasomal or lysosomal digestion of the target of interest. References 1. Dib-Hajj, S. D. et al. Transfection of rat or mouse neurons by biolistics or electroporation. Nat. Protoc.4, 1118–1127 (2009). 2. Faber, C. G. et al. Gain-of-function Nav1.8 mutations in painful neuropathy. Proc. Natl. Acad. Sci. U. S. A.109, 19444–19449 (2012). 3. Baker, C. A. et al. Paclitaxel effects on axonal localization and vesicular trafficking of NaV1.8. Front. Mol. Neurosci.16, (2023). 4. Milescu, L. S., Bean, B. P. & Smith, J. C. Isolation of Somatic Na+ Currents by Selective Inactivation of Axonal Channels with a Voltage Prepulse. J. Neurosci.30, 7740–7748 (2010). 5. Han, C. et al. Human Nav1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons. J. Neurophysiol.113, 3172–3185 (2015). 6. Ghovanloo, M.-R. et al. Sodium currents in naïve mouse dorsal root ganglion neurons: No major differences between sexes. Channels 18, 2289256 (2024). 7. Akin, E. J. et al. Building sensory axons: Delivery and distribution of NaV1.7 channels and effects of inflammatory mediators. Sci. Adv.5, eaax4755 (2019). 8. Higerd-Rusli, G. P. et al. Inflammation differentially controls transport of depolarizing Nav versus hyperpolarizing Kv channels to drive rat nociceptor activity. Proc. Natl. Acad. Sci. 120, e2215417120 (2023). 9. Higerd-Rusli, G. P. et al. Depolarizing NaV and Hyperpolarizing KV Channels Are Co- Trafficked in Sensory Neurons. J. Neurosci.42, 4794–4811 (2022). 10. Tyagi, S. et al. Conserved but not critical: Trafficking and function of NaV1.7 are independent of highly conserved polybasic motifs. Front. Mol. Neurosci.16, (2023). 11. Tyagi, S. et al. Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-α. Cell Rep.43, 113685 (2024). 12. Dib-Hajj, S. D. et al. Transfection of rat or mouse neurons by biolistics or electroporation. Nat. Protoc.4, 1118–1127 (2009). 13. Liu, C. et al. CAP-1A is a novel linker that binds clathrin and the voltage-gated sodium channel Nav1.8. Mol. Cell. Neurosci.28, 636–649 (2005). 14. Rochon, J., Gondan, M. & Kieser, M. To test or not to test: Preliminary assessment of normality when comparing two independent samples. BMC Med. Res. Methodol.12, 81 (2012).45678892.199 ATTORNEY DOCKET NO. YU 8723 PCT 15. Laedermann, C. J. et al. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain. J. Clin. Invest.123, 3002–3013 (2013). 16. Gasser, A. et al. Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK. J. Biol. Chem.285, 26149–26161 (2010). 17. Edwin, F., Anderson, K. & Patel, T. B. HECT Domain-containing E3 Ubiquitin Ligase Nedd4 Interacts with and Ubiquitinates Sprouty2. J. Biol. Chem.285, 255–264 (2010). 18. Foot, N., Henshall, T. & Kumar, S. Ubiquitination and the Regulation of Membrane Proteins. Physiol. Rev.97, 253–281 (2017). 19. Morgenstern, T. J., Park, J., Fan, Q. R. & Colecraft, H. M. A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits. eLife 8, e49253 (2019). 20. Morgenstern, T. J. et al. Selective posttranslational inhibition of CaVβ1-associated voltage-dependent calcium channels with a functionalized nanobody. Nat. Commun.13, 7556 (2022). 21. Dubin, A. E. & Patapoutian, A. Nociceptors: the sensors of the pain pathway. J. Clin. Invest.120, 3760–3772 (2010). 22. Ozaktay, A. C. et al. Effects of interleukin-1 beta, interleukin-6, and tumor necrosis factor on sensitivity of dorsal root ganglion and peripheral receptive fields in rats. Eur. Spine J. Off. Publ. Eur. Spine Soc. Eur. Spinal Deform. Soc. Eur. Sect. Cerv. Spine Res. Soc.15, 1529– 1537 (2006). 23. Chen, X. et al. TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury. Exp. Neurol.227, 279–286 (2011). 24. Spicarova, D., Nerandzic, V. & Palecek, J. Modulation of spinal cord synaptic activity by tumor necrosis factor α in a model of peripheral neuropathy. J. Neuroinflammation 8, 177 (2011). 25. Mo, H. et al. Association of Taxane Type With Patient-Reported Chemotherapy-Induced Peripheral Neuropathy Among Patients With Breast Cancer. JAMA Netw. Open 5, e2239788 (2022). 26. Flatters, S. J. L., Dougherty, P. M. & Colvin, L. A. Clinical and preclinical perspectives on Chemotherapy-Induced Peripheral Neuropathy (CIPN): a narrative review. Br. J. Anaesth. 119, 737–749 (2017). 27. Akin, E. J. et al. Paclitaxel increases axonal localization and vesicular trafficking of Nav1.7. Brain 144, 1727–1737 (2021).45678892.1100 ATTORNEY DOCKET NO. YU 8723 PCT 28. Alsaloum, M., Higerd, G. P., Effraim, P. R. & Waxman, S. G. Status of peripheral sodium channel blockers for non-addictive pain treatment. Nat. Rev. Neurol.16, 689–705 (2020). 29. Bennett, D. L., Clark, A. J., Huang, J., Waxman, S. G. & Dib-Hajj, S. D. The Role of Voltage-Gated Sodium Channels in Pain Signaling. Physiol. Rev.99, 1079–1151 (2019). 30. Goodwin, G. & McMahon, S. B. The physiological function of different voltage-gated sodium channels in pain. Nat. Rev. Neurosci.22, 263–274 (2021). 31. Catterall, W. A., Kalume, F. & Oakley, J. C. NaV1.1 channels and epilepsy. J. Physiol. 588, 1849–1859 (2010). 32. Yu, F. H. & Catterall, W. A. Overview of the voltage-gated sodium channel family. Genome Biol.4, 207 (2003). 33. Li, Z. et al. Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome. Proc. Natl. Acad. Sci.118, e2100069118 (2021). 34. Sizova, D. V. et al. A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane. J. Biol. Chem.295, 1077–1090 (2020). 35. Waxman, S. G. Sodium channels, the electrogenisome and the electrogenistat: lessons and questions from the clinic. J. Physiol.590, 2601–2612 (2012). 36. Bourinet, E. et al. Calcium-Permeable Ion Channels in Pain Signaling. Physiol. Rev.94, 81–140 (2014). 37. Tsantoulas, C. & McMahon, S. B. Opening paths to novel analgesics: the role of potassium channels in chronic pain. Trends Neurosci.37, 146–158 (2014). 38. Ghovanloo, M.-R. et al. High-throughput combined voltage-clamp / current-clamp analysis of freshly isolated neurons. Cell Rep. Methods 3, 100385 (2023). 39. Jones, J. et al. Selective Inhibition of NaV1.8 with VX-548 for Acute Pain. N. Engl. J. Med.389, 393–405 (2023). 40. Wallace, M. S. Trials for Managing Acute Pain — A Clinically Meaningful Small Effect Size? N. Engl. J. Med.389, 464–465 (2023). 41. Waxman Stephen G. Targeting a Peripheral Sodium Channel to Treat Pain. N. Engl. J. Med.389, 466–469 (2023). 42. Chandran, J. et al. Assessment of AAV9 distribution and transduction in rats after administration through Intrastriatal, Intracisterna magna and Lumbar Intrathecal routes. Gene Ther.30, 132–141 (2023).45678892.1101 ATTORNEY DOCKET NO. YU 8723 PCT 43. Gray, S. J., Nagabhushan Kalburgi, S., McCown, T. J. & Jude Samulski, R. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther.20, 450–459 (2013). 44. Flotte, T. R. et al. AAV gene therapy for Tay-Sachs disease. Nat. Med.28, 251–259 (2022). 45. Daci, R. & Flotte, T. R. Delivery of Adeno-Associated Virus Vectors to the Central Nervous System for Correction of Single Gene Disorders. Int. J. Mol. Sci.25, 1050 (2024). 46. Hinderer, C. et al. Severe Toxicity in Nonhuman Primates and Piglets Following High- Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN. Hum. Gene Ther.29, 285–298 (2018). 47. Hudry, E. et al. Liver injury in cynomolgus monkeys following intravenous and intrathecal scAAV9 gene therapy delivery. Mol. Ther.31, 2999–3014 (2023). 48. Muhuri, M., Levy, D. I., Schulz, M., McCarty, D. & Gao, G. Durability of transgene expression after rAAV gene therapy. Mol. Ther.30, 1364–1380 (2022). 49. Gray, S. J. et al. Optimizing Promoters for Recombinant Adeno-Associated Virus- Mediated Gene Expression in the Peripheral and Central Nervous System Using Self- Complementary Vectors. Hum. Gene Ther.22, 1143–1153 (2011). 50. Solé, L. & Tamkun, M. M. Trafficking mechanisms underlying Nav channel subcellular localization in neurons. Channels 14, 1–17 (2020). 51. Stajković, N. et al. Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry. J. Cell Sci.136, jcs260600 (2023). 52. Higerd-Rusli, G. P. et al. The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation. J. Biol. Chem.299, (2023). 53. Lee, J.-H. et al. A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief. Cell 157, 1393–1404 (2014). 54. Atkins, J. F. et al. A case for ‘StopGo’: reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go). RNA 13, 803–810 (2007). 55. Luke, G. A. et al. Occurrence, function and evolutionary origins of ‘2A-like’ sequences in virus genomes. J Gen Virol 89, 1036–1042 (2008). 56. Cox, J. J. et al. An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894–898 (2006).45678892.1102 ATTORNEY DOCKET NO. YU 8723 PCT It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.45678892.1103

Claims

ATTORNEY DOCKET NO. YU 8723 PCT CLAIMS We claim:

1. A fusion polypeptide or a functional fragment or variant thereof and an optional heterologous sequence, optionally packaged in or otherwise associated with a delivery vehicle, wherein the fusion polypeptide is represented by the Formula I, D1-L1-D2, where D1 is a first bioactive domain, D2 is a second bioactive domain, and L1 is an optional flexible linker, wherein the first or second bioactive domains comprise a domain with selective binding to Nav1.8 channels and domain with either ubiquitin ligase activity, ubiquitin ligase recruitment activity or lysosome recruitment activity.

2. The polypeptide of claim 1, wherein domain with ubiquitin ligase activity comprises HECT (homologous to the E6-AP C-terminus) of NEDD4 and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1).

3. The polypeptide of claim 1, wherein the domain with ubiquitin ligase activity comprises a RING (Really Interesting New Gene) domain and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1).

4. The polypeptide of claim 1, wherein the domain with ubiquitin ligase recruitment activity comprises a degron peptide and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1).

5. The polypeptide of claim 1, wherein the domain with lysosome recruitment activity comprises SEQ ID NO: 35 or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35 and the domain with selective binding to Nav1.8 channels comprises the Myosin tail domain (MTD) of clathrin linker-1 (SCLT-1).

6. The polypeptide of claim 5, wherein the wherein the domain with lysosome recruitment activity comprises SEQ ID NO:

35.

7. The polypeptide of any one of claims 1-6, wherein D1 or D2 comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,.45678892.1104ATTORNEY DOCKET NO. YU 8723 PCT 8. The polypeptide of any one of claims 1-7, wherein D1 or D2 comprises SEQ ID NO:5 or a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

5.

9. The polypeptide of any one of claims 1-8, comprising SEQ ID NO:2 or 3 and / or SEQ ID NO:

5.

10. The polypeptide of any one of claims 1-8, comprising SEQ ID NO:26 and / or SEQ ID NO:

5.

11. The polypeptide of any one of claims 1-8, comprising SEQ ID NO:27 and / or SEQ ID NO:

5.

12. The polypeptide of any one of claims 1-8, comprising SEQ ID NO:28 and / or SEQ ID NO:

5.

13. The polypeptide of any one of claims 1-8, comprising SEQ ID NO:29 and / or SEQ ID NO:

5.

14. The polypeptide of any one of claims 1-13, wherein L1 is selected from the group consisting of SEQ ID NO: 7-12.

15. The polypeptide of any one of claims 1-14, wherein D1 comprises SEQ ID NO:2 and D2 comprises SEQ ID NO:

5.

16. The polypeptide of any one of claims 1-14, wherein D1 comprises SEQ ID NO:26 and D2 comprises SEQ ID NO:

5.

17. The polypeptide of any one of claims 1-14, wherein D1 comprises SEQ ID NO:27 and D2 comprises SEQ ID NO:

5.

18. The polypeptide of any one of claims 1-14, wherein D1 comprises SEQ ID NO:28 and D2 comprises SEQ ID NO:

5.

19. The polypeptide of any one of claims 1-14, wherein D1 comprises SEQ ID NO:29 and D2 comprises SEQ ID NO:

5.

20. The polypeptide of any one of claims 1-19, comprising a heterologous sequence, wherein the heterologous sequence comprises one or more of a protein transduction domain, fusogenic polypeptide, targeting signal, expression and / or purification tag.

21. The polypeptide of any one of claims 1-20, comprising a delivery agent.

22. The polypeptide of any one of claims 1-21, wherein the polypeptide can interact with selective binding to Nav1.8 channels.

23. The polypeptide of any one of claims 1-20, comprising a mutated PEST motif with reduce activity.45678892.1105ATTORNEY DOCKET NO. YU 8723 PCT 24. The polypeptide of any one of claims 1-23, wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

5.

25. The polypeptide of any one of claims 1-24, wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO 2.

26. The polypeptide of any one of claims 1-25 wherein D1 or D2 comprises a functional fragment or variant thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

3.

27. A nucleic acid comprising a nucleic acid encoding the polypeptide of any one of claims 1-26, optionally packaged in a delivery vehicle.

28. The nucleic acid of claim 27 comprising SEQ ID NO:

1.

29. The nucleic acid of any one of claims 27 or 28, wherein the nucleic acid is RNA or DNA.

30. The nucleic acid of any one of claims 27-29, wherein the nucleic acid comprises an expression control sequence(s).

31. The nucleic acid of any one of claims 27-30, wherein the nucleic acid is a vector.

32. The nucleic acid of claim 31, wherein the nucleic acid is a viral vector.

32. The nucleic acid of any one of claims 27-30, wherein the nucleic acid is mRNA.

33. The nucleic acid of any one of claims 27-32, wherein the nucleic acid comprises a promotor.

34. The nucleic acid of claim 33, wherein the promotor is a neuron-specific promoter.

35. The nucleic acid of claim 34, wherein the neuron-specific promoter is selected from the group consisting of t Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, neuron-specific enolase (NSE) promoter, and synapsin I with a minimal CMV sequence (SynI- minCMV) promoter.

36. The nucleic acid of any one of claims 27-35 comprising one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.

37. The nucleic acid of any one of claims 32-36, wherein the viral vector is an Adeno-associated virus (AAV)- 38. The nucleic acid of any one of claims 27-37 comprising the delivery vehicle.

39. The polypeptide of any one of claims 1-26 or nucleic acid of any one of claims 27-38, wherein the delivery vehicle is formed of polymeric particles, inorganic particles, silica particles, liposomes, micelles, or multilamellar vesicles, optionally wherein the delivery vehicles45678892.1106ATTORNEY DOCKET NO. YU 8723 PCT comprise one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.

40. A pharmaceutical composition comprising the polypeptide of any one of claims 1-26 or nucleic acid of any one of claims 27-38 alone or packaged in a delivery vehicle optionally formed from formed of polymeric particles, inorganic particles, silica particles, liposomes, micelles, or multilamellar vesicles, optionally wherein the delivery vehicles comprise one or more of a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.

41. A method of treating a subject in need thereof comprising administering the subject an effective amount of the pharmaceutical composition of claim 40.

42. The method of claim 41, wherein the subject has acute pain.

43. The method of claim 42, wherein the subject has chronic pain 44. The method of any one of claims 41-43, wherein the pain results from a condition selected from the group consisting of diabetes, arthritis, fibromyalgia, irritable bowel syndrome, back pain, surgery, cancer, migraines, neurogenic pain, and orchialgia.

45. The method of any one of claims 41-44, wherein the pharmaceutical composition comprises the fusion protein of Formula I.

46. The method of any of any one of claims 41-44, wherein the pharmaceutical composition comprises a nucleic acid encoding the fusion protein of Formula I.

47. The method of any of any one of claims 41-44, wherein the pharmaceutical composition comprises a nucleic acid encoding the fusion protein of Formula I, in a vector.

48. The method of claim 45, wherein the vector is an adeno associated virus (AAV) capsid.

49. The method of claim 48, wherein the AAV capsid is selected from the group consisting of AAV1, AAV4, AAV5, AAV6, AAV8, and AAV9.

50. The method of any one of claims 41-49, wherein the pharmaceutical composition is delivered intrathecally.

51. The method of any one of claims 41-50, wherein the pharmaceutical composition is delivered via injection or infusion 52. The method of one of claims 41-51, wherein the pharmaceutical composition is effective to reduce expression of Nav1.8 channels in the subject.45678892.1107

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