Compositions and methods for using engineered deubiquitinases for targeted stabilization of voltage-gated sodium channels

Abstract

The present disclosure provides, inter alia, an engineered molecule and methods for treating or ameliorating an inherited ion channelopathy, such as Brugada syndrome, or Dravet syndrome, in a subject. Further provided are methods for regulating an ion channel density and ion influx thereof by the engineered molecule disclosed herein.

Description

Docket No. 1035795.000881COMPOSITIONS AND METHODS FOR USING ENGINEERED DEUBIQUITINASES FOR TARGETED STABILIZATION OF VOLTAGE-GATED SODIUM CHANNELSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 708,953, filed on October 18, 2024, the entire content of which is hereby incorporated by reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides, inter alia, an engineered molecule and methods for treating or ameliorating an inherited ion channelopathy, such as Brugada syndrome, or Dravet syndrome, in a subject. Methods for regulating an ion channel density and ion influx thereof by the engineered molecule disclosed above, are also provided herein.GOVERNMENT FUNDING

[0003] This invention was made with government support under grant nos. HL142111 , NS126850, and HL163576, awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0004] This application contains references to amino acids and / or nucleic acid sequences that have been filed concurrently herewith as sequence listing XML file “CU25076-PCT-seq.xml”, file size of 2,129 bytes, created on October 16, 2025. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1 .52(e)(5).BACKGROUND OF THE DISCLOSURE

[0005] Integral surface membrane proteins including ion channels, transporters, and receptors are vital to the survival and function of all cells. Consequently, processes that control the surface abundance and composition of membrane proteinsare critical determinants of cellular biology and physiology. Impaired surface trafficking of membrane proteins underlies diverse diseases ranging from cystic fibrosis to cardiac arrhythmias (Gelman & Kopito, 2002; Anderson et al., 2014), motivating a need to better understand fundamental mechanisms controlling membrane protein surface density. The surface repertoire of membrane proteins is regulated by multilayered maturation and trafficking processes; starting with proper folding in the endoplasmic reticulum (ER), post-translational maturation in the Golgi apparatus, delivery to and continuous refinement at the surface, and ultimately, their removal and degradation in lysosomes (MacGurn et al., 2012; Foot et al., 2017). Basic understanding of the mechanisms that control these diverse aspects of membrane protein fate is an important and intensely studied research area.

[0006] Ubiquitination is a powerful mechanism capable of tuning membrane protein functional expression by regulating multiple steps in the membrane protein lifecycle. Ubiquitin is a 76-residue protein that can be covalently attached to lysine residues on polypeptide substrates through the sequential action of three enzymes: a ubiquitin activation enzyme (E1 ); a ubiquitin-conjugating enzyme (E2); and a ubiquitin ligase (E3), that catalyzes transfer of ubiquitin to substrates. The human genome encodes 2 E1 s, 37 E2s, and >600 E3 ubiquitin ligases. Ubiquitin contains seven lysine residues (K6, K11 , K27, K29, K33, K48, K63) that, together with its N-terminus methionine (Met1 ), can serve as secondary attachment points to make diverse polyubiquitin chains with different structures and functions (Komander, 2009). Ubiquitination has classically been ascribed to targeting cytosolic proteins for degradation by the proteasome (Hershko & Ciechanover, 1998). In contrast, ubiquitination of membrane proteins can lead to more nuanced outcomes including regulating protein trafficking / sorting, stability, and / or function (Komander, 2009; Foot et al., 2017). Ubiquitination has been associated with inherited disorders such as cystic fibrosis, cardiac arrhythmias, epilepsy, and neuropathic pain, as well as infectious disease, contributing to the pathogenic lifecycle of diverse viral and bacterial pathogens.

[0007] Deubiquitinases (DUBs) are specialized isopeptidases that provide salience to ubiquitin signaling through the revision and removal of ubiquitin chains. There are over 100 human DUBs, comprising 6 distinct families: 1 ) the ubiquitin specific proteases (USP) family, 2) the ovarian tumor proteases (OUT) family, 3) the ubiquitin C-terminal hydrolases (UCH) family, 4) the Josephin domain (Josephin) family, 5) the motif interacting with ubiquitin-containing novel DUB (MINDY) family, and 6) theJAB1 / MPN / Mov34 metalloenzyme domain (JAMM) family. Of note, the USP family is relatively promiscuous, hydrolyzing all ubiquitin linkages, in stark contrast to the OTU family, which contains a diverse set of enzymes with distinct linkage preferences. Linkage-specific DUBs have recently been purified and used in cell-free in vitro assays as a way to diagnose chain specificity before running target proteins on Western blot. Moreover, purified linkage-specific DUBs have been used to sculpt more atypical ubiquitin chains for isolation and structural analysis.

[0008] Inherited ion channelopathies are rare diseases that encompass a broad range of disorders in the nervous system (epilepsy, migraine, neuropathic pain), cardiovascular system (long QT syndrome, Brugada syndrome, Dravet syndrome), respiratory (cystic fibrosis), endocrine (diabetes, hyperinsulinemic hypoglycemia), and urinary (Bartter syndrome, diabetes insipidus) system. The rapidly expanding field of next generation genomic sequencing has revealed thousands of channel mutations, with diverse underlying mechanisms. Understanding the underlying cause of loss-of- function is critical for employing a personalized strategy to treat each disease. A vast number of these inherited mutations result in channels with defects in trafficking to the surface membrane. For example, cystic fibrosis, the most common lethal genetic disease in Caucasians arises due to defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel. The most studied mutation (AF508), accounts for -85% of all cases, and causes channel misfolding and ubiquitindependent trafficking defects. In the case of Long QT Syndrome, over 500 mutations in two channels (KCNQ1 , hERG) encompass nearly 90% of all inherited cases. Trafficking deficits in the two channels is the mechanistic basis for a majority of the disease-causing mutations. It would be beneficial to have a platform for high- throughput screening of such disease-causing mutations to diagnose underlying pathological mechanisms. This diagnostic capability would inform on the best treatment options for particular mutations, thereby advancing personalized medicine for these rare diseases. The disclosure provided herein relates to such platform. In addition, the disclosure provides a novel therapeutic opportunity for gene therapy and targeted correction of ubiquitin-dependent trafficking defects that are uncovered.SUMMARY OF THE DISCLOSURE

[0009] The present disclosure provides an engineered molecule comprising: a) a catalytic unit comprising (i) a catalytic domain of a deubiquitinase, or(ii) a targeting domain comprising an antigen-binding domain, that binds to a deubiquitinase; and b) a protein binder comprising an antigen-binding domain, that specifically binds a voltage-gated sodium channel for deubiquitination by the engineered molecule.

[0010] The present disclosure also provides a nucleic acid sequence that encodes the engineered molecule disclosed herein, a recombinant expression vector comprising said nucleic acid sequence, and further provides a cell transformed with said vector.

[0011] The present disclosure also provides a composition comprising the engineered molecule, the nucleic acid sequence, the recombinant expression vector, or the cell disclosed herein, with a pharmaceutically acceptable carrier.

[0012] The present disclosure also provides a method for regulating an ion channel density and ion influx thereof in a subject, comprising administering to the subject a therapeutically effective amount of the engineered molecule, the nucleic acid sequence, the recombinant expression vector, the cell, or the composition disclosed herein.

[0013] The present disclosure also provides a method of treating or ameliorating the effects of an inherited ion channelopathy in a subject, comprising administering to the subject therapeutically effective amount of the engineered molecule, the nucleic acid sequence, the recombinant expression vector, the cell, or the composition disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIGS. 1A-1 F show that GFP nanobody (gnano) linked deubiquitinases increase Nav1.1 -Venus currents.

[0016] FIG. 1A is a cartoon schematic of membrane protein lifecycle. enDUB increases lifespan of membrane protein by reducing protein degradation through the ubiquitin-proteasome pathway.

[0017] FIG. 1 B is a channel schematic of Nav1.1 -Venus targeted by a GFP nanobody (gnano) on the C-terminus (left). Exemplar family of whole-cell electrophysiology current traces from HEK293 cells transiently transfected with NaV1 .1 -Venus. Cells were held at -120 mV and given voltage steps from -80 mV to +90 mV in +10 mV increments for 20 ms (right).

[0018] FIG. 1 C shows that OTUD1 conjugated to gnano (gnano-OTUD1 ) greatly enhances NaV1.1 -Venus currents. Format is the same as FIG. 1 B.

[0019] FIG. 1 D shows that OTUD4 conjugated to gnano (gnano-OTUD4) does not affect NaV1.1-Venus currents. Format is the same as FIG. 1 B.

[0020] FIG. 1 E shows the population summary of voltage-current relationship for peak current density (Ipeak) shown as average ± SEM: gnano (n=7), gnano-OTUD1 (n=7), gnano-OTUD4 (n=11).

[0021] FIG. 1 F shows the population summary of voltage dependence of inactivation (Norm Finact) shown as average ± SEM: gnano (n=6), gnano-OTUD1 (n=7), gnano-OTUD4 (n=10). Transfected cells were held at -120 mV then given voltage steps from -140 mV to 0 mV in +10 mV increments for 100 ms before stepping to a test voltage of -20 mV for 20 ms. Peak current recorded at the test voltage is divided by the peak current from the voltage step to yield the normalized fraction of inactivation.

[0022] FIGS. 2A-2E show the targeted enhancement of Nav1.1 current with nb82 linked deubiquitinases.

[0023] FIG. 2A shows that whole-cell electrophysiology of HEK293 cells stably expressing NaV1.1 show robust sodium currents with nb82. Format is the same as FIG. 1 B.

[0024] FIG. 2B shows that the addition of nb82-OTUD1 significantly increases sodium currents. Format is the same as FIG. 1 B.

[0025] FIG. 2C shows that mutating the catalytic site of OTUD1 shows NaV1.1 current is no longer increased by enDUB. Format is the same as FIG. 1 B.

[0026] FIG. 2D shows the population summary of voltage-current relationship for peak current density (Ipeak) shown as average ± SEM: nb82 (n=10), nb82-OTUD1 (n=11 ), nb82-OTUD1 (C320S) catalytically dead (n=8). Format is the same as FIG. 1 E.

[0027] FIG. 2E shows the population summary of voltage dependence of inactivation (Norm Finact) shown as average ± SEM: nb82 (n=9), nb82-OTUD1 (n=9),nb82-OTUD1 (C320S) catalytically dead (n=8). Format is the same as FIG. 1 F.

[0028] FIGS. 3A-3E show the targeted enhancement of Nav1.5 current with nanobody-targeted deubiqutinases.

[0029] FIG. 3A shows that exemplar traces showing whole cell Nav1.5 currents heterologously expressed in HEK293 cells evoked in response to a family of voltagesteps from -120 mV to +50 mV with a holding potential of -120 mV.

[0030] FIG. 3B shows that co-expression of nb82-targed OTUD1 deubiquitinase upregulates peak Na current. A previously established FixR peptide was attached to the amino-terminus of nb82 to enhance inactivation and prevent any change in late Na current which can be proarrhythmic.

[0031] FIG. 3C shows that co-expression of nb82-targeted OTUD4 resulted in a reduction in peak Nav1.5 current.

[0032] FIG. 3D shows that co-expresssion of nb82-targeted USP21 also caused a reduction in peak Nav1.5 current.

[0033] FIG. 3E shows the population data that summarizes changes in peak NaV1.5 current density with various nb82-targetted deubiquitinases. Only OTUD1 caused a significant upregulation of peak current density.

[0034] FIGS. 4A-4C shows the targeted upregulation of loss-of-fucntion Navi .5 variants by nanobody-fused OTUD1 .

[0035] FIG. 4A shows that Navi .5 E1784K variant linked to both long-QT syndrome type 3 and Brugada Syndrome shows a baseline reduction in peak Na current. Coexpression of nanobody-targeted OTIID1 reverses this deficit in peak current density. Top, population data. Bottom, whole-cell exemplary traces.

[0036] FIG. 4B shows that Navi .5 Q1909R variant also shows a baseline reduction in peak Na current. Overexpression of nanobody-targeted OTLID1 results in a marked increase in peak current density.

[0037] FIG. 4C shows that Nav1.5 S1904L variant also exhibits reduced peak current density which is upregulated by nanobody-targeted OTUD1.

[0038] FIGS. 5A-5C show that nanobody-fused OTLID1 can reverse deficits in peak Na current density in a mouse model for heart failure with preserved ejection fraction (HFpEF).

[0039] FIG. 5A shows a previously established 2-hit model of HFpEF resulting from obesity due to high-fat diet and hypertension resulting from inhibition of nitric-oxide synthase activity (L-NAME).

[0040] FIG. 5B shows that compared to mice fed with normal chow diet (WT), mice on high fat diet and L-name (HF) have increased bodyweight.

[0041] FIG. 5C shows that isolated cardiomyocytes from HF mice show a significant reduction in peak Na current compared to mice on a normal diet regiment (WT). Retro-oribital injection of AAX / 9 encoding nanobody-fused OTUD1 resulted in robust expression and functional rescue of peak Na current in cardiac myocytes isolated from HF model.DETAILED DESCRIPTION OF THE DISCLOSURE

[0042] The present disclosure provides a genetic tool for addressing human neuronal and cardiac diseases such as Dravet and Brugada Syndrome, which are caused by de novo or inherited genetic mutations in voltage-gated sodium channels Nav1.1 and Nav1.5 respectively. More specifically, genetic mutations in voltage-gated sodium channels associated with these diseases are mechanistically identified as loss-of-function where there is a reduction in sodium ion influx during channel activation resulting in loss of electrical signaling in the brain and the heart. Beyond genetic diseases, pathological remodeling as a result of heart failure causes a reducition in Nav1.5 current, which is thought to contribute to increased risk of arrhythmias and mortality in this patient population. The present disclosure specifically targets a deubiquitinase (e.g., OTLID1 ) to its respective channel with a nanobody to stabilize channel expression on the plasma membrane of the cell, thus upregulating channel density and restoring sodium ion influx into the cell. Current technologies for addressing these diseases include drugs for symptom management and targeted augmentation of nuclear gene output (TANGO) using anti-sense oligos (ASOs). Drugs on the market do not solve the underlying deficits and only mitigate patient symptoms, if at all. Unlike TANGO, the present disclosure uses a post-translational mechanism to increase the surface density of WT Navi channels. The present disclosure provides not only targeted specificity to voltage-gated sodium channels, Navi .1 and Navi .5 but also generalizability for stabilizing and upregulating sodium channels on the plasma membrane of cells so that one wildtype allele in a patient is sufficient for ion influx rescue.

[0043] One aspect of the present disclosure relates to an engineered molecule comprising:a) a catalytic unit comprising (i) a catalytic domain of a deubiquitinase, or (ii) a targeting domain comprising an antigen-binding domain, that binds to a deubiquitinase; and b) a protein binder comprising an antigen-binding domain, that specifically binds a voltage-gated sodium channel for deubiquitination by the engineered molecule.

[0044] In some embodiments, the catalytic unit and the protein binder are operably connected by a variable linker. As used herein, a “variable linker” refers to a molecule that can be used to connect or bridge different parts of a protein or different proteins, or to link a protein or parts of it to another molecule or surface. The variable linker can be either inorganic (i.e., non-carbon-based) or a segment of amnio acids (peptide). The length of the variable linker can be changed to control the distance and flexibility between two or more protein domains orfunctional units, maintaining the independent function of each domain / unit while allowing them to work together.

[0045] In some embodiments, the catalytic unit of the engineered molecule is non- selective for a particular ubiquitin linkage type. In some other embodiments, the catalytic unit is selective for a particular ubiquitin linkage type.

[0046] In some embodiments, the catalytic unit comprises the catalytic domain of a deubiquitinase selected from the ubiquitin specific proteases (USP) family, the ovarian tumor proteases (OTU) family, the ubiquitin C-terminal hydrolases (UCH) family, the Josephin domain (Josephin) family, the motif interacting with ubiquitin- containing novel DUB (MINDY) family, or the JAB1 / MPN / Mov34 metalloenzyme domain (JAMM) family.

[0047] In some embodiments, the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family. In one embodiment, the catalytic unit comprises the catalytic domain of USP21 .

[0048] According to some embodiments, the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family. In one embodiment, the catalytic unit comprises the catalytic domain of OTUD1. In one embodiment, the catalytic unit comprises the catalytic domain of OTUD4. In one embodiment, the catalytic unit comprises the catalytic domain of Cezanne. In one embodiment, the catalytic unit comprises the catalytic domain of TRABID. In one embodiment, the catalytic unit comprises the catalytic domain of OTULIN.

[0049] In certain embodiments, the catalytic unit is the catalytic domain of USP21and is capable of non-selectively eliminating all ubiquitin linkage types. In certain embodiments, the catalytic unit is the catalytic domain of OTUD1 and is capable of selectively eliminating K63 ubiquitin linkage. In certain embodiments, the catalytic unit is the catalytic domain of OTLID4 and is capable of selectively eliminating K48 ubiquitin linkage. In certain embodiments, the catalytic unit is the catalytic domain of Cezanne and is capable of selectively eliminating K11 ubiquitin linkage. In certain embodiments, the catalytic unit is the catalytic domain of TRABID and is capable of selectively eliminating K29 ubiquitin linkages, K33 ubiquitin linkages, or K29 and K33 ubiquitin linkages. In certain embodiments, the catalytic unit is the catalytic domain of OTULIN and is capable of selectively eliminating Met1 ubiquitin linkage. As used herein, “capable of” means that the subject, e.g., the engineered molecule, is fully functional and under the proper conditions, will carry out the stated functions.

[0050] The engineered molecule disclosed herein can function as a bivalent molecule (e.g., as those described in WO2021 / 146386, i.e., the catalytic uint of the engineered molecule does not include any catalytic domain of a deubiquitinase, instead it binds to a deubiquitinase and brings the deubiquitinase to the target protein that is captured by the protein binder of the engineered molecule. Accordingly, the present disclosure also provides an engineered molecule comprising: (a) a catalytic unit comprising a targeting domain comprising an antigen-binding domain, that binds to a deubiquitinase; and (b) a protein binder comprising an antigen-binding domain, that specifically binds a voltage-gated sodium channel for deubiquitination by the deubiquitinase captured by the targeting domain in the catalytic unit of the engineered molecule. For example, in one embodiment of the present disclosure, the catalytic unit of the engineered molecule comprises the targeting domain that binds to a deubiquitinase. In certain embodiments, the deubiquitinase captured by the targeting domain of the catalytic unit can be either endogenous or exogenous.

[0051] As used herein, the term “endogenous” refers to a nucleic acid molecule or polypeptide that is normally (or naturally) expressed in a cell or tissue.

[0052] As used herein, the term “exogenous” refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides.

[0053] In some embodiments, the catalytic unit comprises the targeting domain thatbinds to a deubiquitinase, and wherein the deubiquitinase is from the USP family, the OTU family, the UCH family, the Josephin family, the MINDY family, or the JAMM family.

[0054] In some embodiments, the deubiquitinase is from the USP family. In certain embodiments, the deubiquitinase is USP21.

[0055] In some embodiments, the deubiquitinase is from the OTU family. In certain embodiments, the deubiquitinase is OTUD1. In certain embodiments, the deubiquitinase is OTUD4. In certain embodiments, the deubiquitinase is Cezanne. In certain embodiments, the deubiquitinase is TRABID. In certain embodiments, the deubiquitinase is OTULIN.

[0056] In some embodiments, the antibody of the engineered molecule is a single domain antibody (sdAb), a VHH, a single heavy-chain variable region, scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, diabody, or a DARPin. In some embodiments, the antibody is a single domain antibody (sdAb). In one embodiment, the antibody is nb82 comprising an amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the antibody comprises an amino acid sequence that is substantially identical or homologous to the amino acid sequence set forther in SEQ ID NO: 1 .

[0057] By “substantially identical” or “substantially homologous” is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any of the nucleic acid sequences described herein). For exmaple, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the sequence of the amino acid or nucleic acid used for comparison. In certain embodiments of the present disclosure, the protein binder comprising an antigen-binding domain (e.g., an antibody) can comprise an amino acid sequence that is at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 1.

[0058] Sequence identity can be measured by using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0059] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=#of identical positions / total #of positionsxl OO), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0060] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

[0061] Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the specified sequences disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0062] As used herein, a “voltage-gated sodium channel” or “VGSC” refers to a type of microporous transmembrane protein that is widely distributed on the membranes of excitable cells such as neurons, and it is mainly responsible for the transmembrane transport of Na+. It is regulated by a change in transmembrane voltage. In some embodiments, the voltage-gated sodium channel selected from the group consisting of Navi .1 , Nav1.2, Na 1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Navi .9.

[0063] Other aspects of the present disclosure relate to a nucleic acid sequence that encodes the engineered molecule disclosed herein, a recombinant expression vector comprising said nucleic acid sequence, as well as a cell transformed with said vector. In some embodiments, the cell is mammalian or bacterial.

[0064] Another aspect of the present disclosure relates to a composition comprising the engineered molecule, the nucleic acid sequence, the recombinant expression vector, or the cell disclosed herein, with a pharmaceutically acceptable carrier.

[0065] Still another aspect of the present disclosure relates to a method for regulating an ion channel density and ion influx thereof in a subject, comprising administering to the subject a therapeutically effective amount of the engineered molecule, the nucleic acid sequence, the recombinant expression vector, the cell, or the composition disclosed herein.

[0066] In some embodiments, the ion channel is a voltage-gated sodium channel selected from Navi .1 and Navi .5, and the ion influx is a sodium ion influx.

[0067] As used herein, “regulation” or “regulating” can be either upregulation (upregulating) or downregulation (downregulating) of the expression level of a protein such as, e.g., the surface desity of an ion channel on the plasma membrane of a cell, or the ion influx (current) of such an ion channel, e.g., the Nav1.5 current. In some embodiments, the regulation is to stabilize the ion channel density and restore ion influx thereof, and the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD1. In some other embodiments, the regulation is to reduce the ion influx of the ion channel, and the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD4 or USP21 .

[0068] Yet another aspect of the present disclosure relates to a method of treating or ameliorating the effects of an inherited ion channelopathy in a subject, comprising administering to the subject a therapeutically effective amount of the engineered molecule, the nucleic acid sequence, the recombinant expression vector, the cell, or the composition disclosed herein.

[0069] As used herein, the term “inherited ion channelopathy” refers to rare diseases that encompass a broad range of disorders in the nervous system, cardiovascular system, respiratory system, endocrine system, and urinary system. In the present disclosure, an “inherited ion channelopathy” includes but is not limited to epilepsy, migraine, neuropathic pain, cardiac arrhythmias, long QT syndrome, Brugada syndrome, Dravet syndrome, cystic fibrosis, diabetes, hyperinsulinemic hypoglycemia, Bartter syndrome, and diabetes insipidus. In one embodiment, the inherited ion channelopathy is Brugada syndrome or Dravet syndrome.

[0070] In certain embodiments, the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD1 , and the protein binder of the engineered molecule is nb82 comprising an amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the protein binder of the engineered molecule comprises an amino acid sequence that is at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 1.

[0071] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population may fail to respond or respond inadequately to treatment.

[0072] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject, preferably a human.

[0073] As used herein, "administration," "administering" and variants thereof means introducing a composition, such as a synthetic membrane-receiver complex, or agent into a subject and includes concurrent and sequential introduction of a composition or agent. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration can be carried out by any suitable route.

[0010] As used herein, a "therapeutically effective amount" is an amount sufficient to effect beneficial or desired results. A therapeutically effective amount can be administered in one or more doses. The therapeutically effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form of the drug being administered.

[0011] Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of animal, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of an agent according to the disclosure will be that amount of the agent, which is the lowest dose effective to produce the desired effect. The effective dose of an agent may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0074] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals includeprimates, rats, mice, rabbits, guinea pigs, etc.

[0075] In some aspects of this and other embodiments, the subject is a mammal. Preferably, the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammal is a human. Additional Definitions

[0076] The term "amino acid" means naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. An “amino acid analog” means compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Imino acids such as, e.g., proline, are also within the scope of “amino acid” as used here. An “amino acid mimetic” means a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0077] As used herein, the terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non- naturally occurring amino acid polymers.

[0078] “Nucleic acid" or "oligonucleotide" or "polynucleotide" used herein means at least two nucleotides covalently linked together. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0079] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of basesincluding uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be synthesized as a single stranded molecule or expressed in a cell (in vitro or in vivo) using a synthetic gene. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0080] The nucleic acid may also be an RNA such as an mRNA, tRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), transcriptional gene silencing RNA (ptgsRNA), Piwi-interacting RNA, pri-miRNA, pre- miRNA, micro-RNA (miRNA), or anti-miRNA.

[0081] As used herein, the term “antigen-binding domain” refers to a region of a molecule, that recognizes and binds to a specific antigen. Exemplary molecules comprising an antigen-binding domain include, but not limited to, an antibody and antigen binding fragments thereof, a modified antibody, a chimeric antigen receptor (CAR), an antibody mimetic, etc.

[0082] As used herein, the term "antibody" encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partly or wholly synthetically produced. "Antibody" further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. Use of the term antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primatehuman monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1 )2, Fv, dAb, and Fd fragments, diabodies, nanobodies and antibody-related polypeptides. Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.

[0083] The term "antigen binding fragment" used herein refers to fragments of an intact immunoglobulin, and any part of a polypeptide including antigen binding regions having the ability to specifically bind to the antigen. For example, the antigen binding fragment may be a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, or a scFv fragment, but is not limited thereto. A Fab fragment has one antigen binding site and contains the variable regions of a light chain and a heavy chain, the constantregion of the light chain, and the first constant region CH1 of the heavy chain. A Fab' fragment differs from a Fab fragment in that the Fab' fragment additionally includes the hinge region of the heavy chain, including at least one cysteine residue at the C- terminal of the heavy chain CH1 region. The F(ab')2 fragment is produced whereby cysteine residues of the Fab' fragment are joined by a disulfide bond at the hinge region. A Fv fragment is the minimal antibody fragment having only heavy chain variable regions and light chain variable regions, and a recombinant technique for producing the Fv fragment is well known in the art. Two-chain Fv fragments may have a structure in which heavy chain variable regions are linked to light chain variable regions by a non-covalent bond. Single-chain Fv (scFv) fragments generally may have a dimer structure as in the two-chain Fv fragments in which heavy chain variable regions are covalently bound to light chain variable regions via a peptide linker or heavy and light chain variable regions are directly linked to each other at the C-terminal thereof. The antigen binding fragment may be obtained using a protease (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(ab')2 fragments), and may be prepared by a genetic recombinant technique. A dAb fragment consists of a VH domain. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimmer, trimer or other polymers.

[0084] As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that is engineered to enhance the immune system's ability to target and destroy specific cells, such as cancer cells. A CAR usually comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. It is used in caner treatment, for example, CAR-T cell therapy, by modifying the T cells and enabling modifed T cells to target and destroy tumor cells.

[0085] As used herein, ther term “antibody mimetics” refer to molecules that can bind to antigens similar to antibodies but are not generated by the immune system and have no structural relation to the antibodies. Antibody mimetics are mostly unrelated protein scaffolds consisting of a-helices, p-sheets, or random coils that can bind to specific targets and could be designed to incorporate novel binding sites through common protein engineering strategies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Examples of antibody mimetics include, but not limited to, affibodies, DARPins, anticalins, monobodies (adnectins), knottins, optimers, avimers, fynomers, etc.

[0086] As used herein, the terms “single domain antibody”, “variable heavy-chain (VHH) antibody” and “nanobody” have the same meaning referring to a variable region of a heavy chain of an antibody and construct a single domain antibody consisting of only one heavy chain variable region. For example, in some embodiments, the single domain antibody used as described herein is nb82 comprising the amino acid sequence of“QVQLQESGGGLVQTGGSLRLSCKASGRAFARYDLAWSRQAPGKQREFVASIGVT RNPPYYSGSVKGRFTVSRDNAKETVYLQMNDLKPEDSAVYYCAAKDASVTVATIE DYPYWGRGTQVTVSSENLYFQGHHHHHH” (SEQ ID NO: 1).

[0087] As used herein, the term “variable” refers that certain portions of the variable region in the nanobodies vary in sequences, which forms the binding and specificity of various specific antibodies to their particular antigen.

[0088] As used herein, the term “heavy chain variable region” and “VH” can be used interchangeably.

[0089] As used herein, the terms “variable region” and “complementary determining region (CDR)” can be used interchangeably.

[0090] "Vector" used herein refers to an assembly which is capable of directing the expression of desired protein. The vector must include transcriptional promoter elements which are operably linked to the gene(s) of interest. The vector may be composed of either deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (e.g., a DNA-RNA chimeric). Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may also be incorporated into the vectors described herein.

[0091] As used herein, the terms “cell”, “host cell” or "recombinant host cell" refers to host cells that have been engineered to express a desired recombinant protein. Methods of creating recombinant host cells are well known in the art. For example, see Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), Ausubel et al. (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Ausubel et al., eds., John Wiley & Sons, New York, 1987). In the present disclosure, the hostcells are transformed with the vectors described herein.

[0092] Recombinant host cells as used herein may be any of the host cells used for recombinant protein production, including, but not limited to, bacteria, yeast, insect and mammalian cell lines.

[0093] As used herein, the term "increase," "enhance," "stimulate," and / or "induce" (and like terms) generally refers to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[0094] As used herein, the term "inhibit," "suppress," "decrease," "interfere," and / or "reduce" (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[0095] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0096] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0097] The following examples are provided to further illustrate certain aspects of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.EXAMPLESExample 1GFP nanobody (gnano) linked deubiquitinases increase Nav1.1 -Venus currents

[0098] The nanoDUB constructs, the plasmid vectors carrying those constructs, and low passage human embryonic kidney (HEK293) cells expressing WT or mutant sodium channels were created in a similar manner as described in the U.S. Patent No. 11 ,845,967.

[0099] For sodium channel measurements, whole-cell membrane currents wererecorded at room temperature in HEK293 cells using an EPC-10 patch-clamp amplifier (HEKA Electronics) controlled by the PatchMaster software (HEKA). A coverslip with adherent HEK293 cells was placed on the glass bottom of a recording chamber (0.7- 1 mL in volume) mounted on the stage of an inverted Nikon Eclipse Ti-U microscope. Micropipettes were fashioned from 1 .5 mm thin-walled glass and fire-polished. Pipette resistance was typically 1 .5 MQ when filled with internal solution.

[0100] Data were analyzed off-line using FloJo, PulseFit (HEKA), Microsoft Excel, Origin and GraphPad Prism software. Statistical analyses were performed in Origin or GraphPad Prism using built-in functions. Statistically significant differences between means (P < 0.05) were determined using Student’s t test for comparisons between two groups. Data are presented as means ± s.e.m.

[0101] As shown in FIG. 1A, an engineered DUB (enDUB) increases lifespan of membrane protein by reducing protein degradation through the ubiquitin-proteasome pathway. To probe the effect of enDUB on sodium channel current, a family of wholecell electrophysiology current traces from HEK293 cells transiently transfected with Nav1.1 -Venus were recorded. Cells were held at -120 mV and given voltage steps from -80 mV to +90 mV in +10 mV increments for 20 ms. Compared to the control of GFP nanobody (gnano) (FIG. 1 B), OTUD1 conjugated to gnano (gnano-OTUD1 ) greatly enhanced Nav1.1-Venus currents (FIG. 1 C), while OTUD4 conjugated to gnano (gnano-OTUD4) had no effect on Nav1.1 -Venus currents (FIG. 1 D). Population summary of voltage-current relationship for peak current density ( / peak) and voltage dependence of inactivation (Norm Enact) are shown in FIGS. 1 E and 1 F.Example 2Targeted enhancement of Nav1.1 current with nb82 linked deubiquitinases

[0102] In this Example, catalytic domain of OUTD1 was fused to nb82 nanobody to generate two distinct nanoDUBs — nb82-OTUD1 and nb82-OTUD1 (C320S) (catalytically dead by mutating the catalytic site at C320).

[0103] Following the same protocol in Example 1 , whole-cell electrophysiology of HEK293 cells stably expressing Nav1.1 showed robust sodium currents with nb82 (FIG. 2A). While the addition of nb82-OTUD1 significantly increased sodium currents (FIG. 2B), nanoOTUD1 (C320S) was not able to increase the currents (FIG. 2C). Population summary of voltage-current relationship for peak current density ( / peak) and voltage dependence of inactivation (Norm Enact) are shown in FIGS. 2D and 2E.Example 3Targeted enhancement of Navi .5 current with nanobody-targeted deubiqutinases

[0104] Beside the nanoDUBs described in Example 2, more nanoDUBs were developed, including nb82-OTUD4 and nb82-USP21. A previously established FixR peptide to the amino-terminus of nb82 to enhance inactivation and prevent any change in late sodium current which can be proarrhythmic.

[0105] In this Example, a family of whole-cell electrophysiology current traces from HEK293 cells transiently transfected with Nav1.5 were recorded. Cells were held at - 120 mV and given voltage steps from -120 mV to +50 mV in +10 mV increments.

[0106] Compared to WT Nav1.5 (FIG. 3A), Co-expression of nb82-targed OTUD1 deubiquitinase upregulates peak sodium current (FIG. 3B), while both co-expression of nb82-targeted OTUD4 and co-expresssion of nb82-targeted LISP21 resulted in a reduction in peak Navi .5 current (FIGS. 3C and 3D). Population data showed that only OTUD1 caused a significant upregulation of peak current density (FIG. 3E).Example 4Targeted upregulation of loss-of-fucntion Nav1.5 variants by nanobody-fused OTUD1

[0107] To further explore the sodium channel current upregulation by nanobody- fused OTUD1 , I oss-of-fu notion Nav1.5 variants were generated, including mutations at E1784K (linked to both long-QT syndrome type 3 and Brugada Syndrome), Q1909R, and S1904L.

[0108] The E1784K variant showed a baseline reduction in peak Na current density, which was reversed by co-expression of nanobody-targeted OTUD1 (FIG. 4A). Similar results were obtained in both the Q1909R and S1904L variants, with increase in peak current density at different degrees (FIGS. 4B and 4C).Example 5Nanobody-fused OTUD1 can reverse deficits in peak Na current density in a mouse model for heart failure with preserved ejection fraction (HFpEF)

[0109] In this Example, a previously established 2-hit model of HFpEF resulting from obesity due to high-fat diet and hypertension resulting from inhibition of nitric-oxide synthase activity (L-NAME) were used (FIG. 5A). As shown in FIG. 5B, compared to mice fed with normal chow diet (WT), mice on high fat diet and L-name (HF) had increased bodyweight. While isolated cardiomyocytes from HF mice showed a significant reduction in peak Na current compared to mice on a normal diet regiment (WT), retro-oribital injection of AAV9 encoding nanobody-fused OTUD1 resulted in robust expression and functional rescue of peak Na current in cardiac myocytes isolated from HF model (FIG. 50).

[0110] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety as if recited in full herein.

[0111] The disclosure being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the disclosure and all such modifications are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An engineered molecule comprising: a) a catalytic unit comprising (i) a catalytic domain of a deubiquitinase, or (ii) a targeting domain comprising an antigen-binding domain, that binds to a deubiquitinase; and b) a protein binder comprising an antigen-binding domain, that specifically binds a voltage-gated sodium channel for deubiquitination by the engineered molecule.

2. The engineered molecule of claim 1 , wherein the catalytic unit and the protein binder are operably connected by a variable linker.

3. The engineered molecule of claim 1 , wherein the catalytic unit is non-selective for a particular ubiquitin linkage type.

4. The engineered molecule of claim 1 , wherein the catalytic unit is selective for a particular ubiquitin linkage type.

5. The engineered molecule of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase, wherein the deubiquitinase is from the ubiquitin specific proteases (USP) family, the ovarian tumor proteases (OTU) family, the ubiquitin C-terminal hydrolases (UCH) family, the Josephin domain (Josephin) family, the motif interacting with ubiquitin-containing novel DUB (MINDY) family, or the JAB1 / MPN / Mov34 metalloenzyme domain (JAMM) family.

6. The engineered molecule of claim 5, wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family.

7. The engineered molecule of claim 6, wherein the catalytic unit comprises the catalytic domain of USP21 .

8. The engineered molecule of claim 5, wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family.

9. The engineered molecule of claim 8, wherein the catalytic unit comprises the catalytic domain of OTUD1 .

10. The engineered molecule of claim 8, wherein the catalytic unit comprises the catalytic domain of OTUD4.

11. The engineered molecule of claim 8, wherein the catalytic unit comprises the catalytic domain of Cezanne.

12. The engineered molecule of claim 8, wherein the catalytic unit comprises the catalytic domain of TRABID.

13. The engineered molecule of claim 8, wherein the catalytic unit comprises the catalytic domain of OTULIN.

14. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of USP21 and is capable of non-selectively eliminating all ubiquitin linkage types.

15. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of OTUD1 and is capable of selectively eliminating K63 ubiquitin linkage.

16. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of OTUD4 and is capable of selectively eliminating K48 ubiquitin linkage.

17. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of Cezanne and is capable of selectively eliminating K11 ubiquitin linkage.

18. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of TRABID and is capable of selectively eliminating K29 ubiquitin linkages, K33 ubiquitin linkages, or K29 and K33 ubiquitin linkages.

19. The engineered molecule of claim 1 , wherein the catalytic unit is the catalytic domain of OTULIN and is capable of selectively eliminating Met1 ubiquitin linkage.

20. The engineered molecule of claim 1 , wherein the catalytic unit comprises the targeting domain that binds to a deubiquitinase, and whererin the deubiquitinase is endogenous.

21. The engineered molecule of claim 1 , wherein the catalytic unit comprises the targeting domain that binds to a deubiquitinase, and wherein the deubiquitinase is from the USP family, the OTU family, the UCH family, the Josephin family, the MINDY family, or the JAMM family.

22. The engineered molecule of claim 21 , wherein the deubiquitinase is from the USP family.

23. The engineered molecule of claim 22, wherein the deubiquitinase is USP21 .

24. The engineered molecule of claim 21 , wherein the deubiquitinase is from the OTU family.

25. The engineered molecule of claim 24, wherein the deubiquitinase is OTLID1 .

26. The engineered molecule of claim 24, wherein the deubiquitinase is OTUD4.

27. The engineered molecule of claim 24, wherein the deubiquitinase is Cezanne.

28. The engineered molecule of claim 24, wherein the deubiquitinase is TRABID.

29. The engineered molecule of claim 24, wherein the deubiquitinase is OTULIN.

30. The engineered molecule of claim 1 , wherein the antigen-binding domain is selected from the group consisting of an antibody and antigen binding fragments thereof, a modified antibody, a chimeric antigen receptor (CAR), or an antibody mimetic.

31. The engineered molecule of claim 30, wherein the antibody is a single domain antibody (sdAb), a VHH, a single heavy-chain variable region, scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, diabody, or a DARPin.

32. The engineered molecule of claim 31 , wherein the antibody is a single domain antibody (sdAb).

33. The engineered molecule of claim 31 , wherein the antibody is nb82 comprising an amino acid sequence set forth in SEQ ID NO: 1.

34. The engineered molecule of claim 31 , wherein the antibody comprises an amino acid sequence that is at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 1.

35. The engineered molecule of claim 1 , wherein the voltage-gated sodium channel is selected from the group consisting of Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9.

36. A nucleic acid sequence encoding the engineered molecule according to any one of claims 1-35.

37. A recombinant expression vector comprising the nucleic acid sequence of claim 36.

38. A cell transformed with the vector of claim 37.

39. The cell of claim 38, wherein the cell is mammalian or bacterial.

40. A composition comprising the engineered molecule according to any one of claims 1 -35, the nucleic acid sequence according to claim 36, the recombinantexpression vector according to claim 37, or the cell according to claim 38 or claim 39, with a pharmaceutically acceptable carrier.

41. A method for regulating an ion channel density and ion influx thereof in a subject, comprising administering to the subject a therapeutically effective amount of the engineered molecule according to any one of claims 1-35, the nucleic acid sequence according to claim 36, the recombinant expression vector according to claim 37, or the cell according to claim 38 or claim 39, or the composition according to claim 40.

42. The method of claim 41 , wherein the ion channel is a voltage-gated sodium channel selected from Nav1.1 and Nav1.5, and the ion influx is a sodium ion influx.

43. The method of claim 41 , wherein the regulation is to stabilize the ion channel density and restore ion influx thereof, and the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD1 .

44. The method of claim 41 , wherein the regulation is to reduce the ion influx of the ion channel, and the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD4 or USP21 .

45. A method of treating or ameliorating the effects of an inherited ion channelopathy in a subject, comprising administering to the subject a therapeutically effective amount of the engineered molecule according to any one of claims 1-35, the nucleic acid sequence according to claim 36, the recombinant expression vector according to claim 37, or the cell according to claim 38 or claim 39, or the composition according to claim 40.

46. The method of claim 45, wherein the subject is mammal.

47. The method of claim 46, wherein the subject is human.

48. The method of claim 45, wherein the inherited ion channelopathy is selected from the group consisting of epilepsy, migraine, neuropathic pain, cardiac arrhythmias, long QT syndrome, Brugada syndrome, Dravet syndrome, cystic fibrosis, diabetes, hyperinsulinemic hypoglycemia, Bartter syndrome, and diabetes insipidus.

49. The method of claim 45, wherein the inherited ion channelopathy is Brugada syndrome or Dravet syndrome.

50. The method of claim 45, wherein the catalytic unit of the engineered molecule comprises the catalytic domain of OTUD1 , and the protein binder of theengineered molecule is nb82 comprising an amino acid sequence set forth in SEQ ID NO: 1.

51. The method of claim 50, wherein the protein binder of the engineered molecule comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 1.

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