Synthetic autophagy receptors and uses thereof

Abstract

The present disclosure relates generally to the field of targeted degradation, and particularly relates to synthetic autophagy receptors, pharmaceutical compositions containing the same, nucleic acid molecules encoding the same, compositions for production of desired products in cell cultures or in a living body, and methods of use.

Description

SYNTHETIC AUTOPHAGY RECEPTORS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 584,617, filed September 22, 2023. The contents of the above-referenced application is herein expressly incorporated by reference in its entirety, including any drawings.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under F32 GM139242. and R01 GM130145 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present disclosure relates generally to compositions and methods comprising synthetic authophagy receptors.BACKGROUND

[0004] Cellular homeostasis requires the degradation of abnormal protein aggregates and damaged organelles. The accumulation of impaired protein aggregates and organelles has been linked with aging and neurodegenerative diseases. For example, tau aggregation is a hallmark of Alzheimer's disease and the aberrant aggregation of TAR DNA-binding protein 43 kDa (TDP-43) has been identified as a defining pathological feature for amyotrophic lateral sclerosis (ALS). The accumulation of defective mitochondria can cause severe cell and tissue damage as observed in patients with Parkinson’s disease. It is thus important to maintain an efficient clearance of unwanted large-sized intracellular contents.

[0005] In living cells, dysfunctional protein aggregates, organelles, and pathogenic microorganisms are degraded through autophagy. Autophagy initiates the degradation of intracellular contents through the formation of the double-membrane autophagosomes, followed by fusion with lysosomes and proteolytic degradation of the engulfed substrates. Unlike the ubiquitin-proteasome system (UPS) where proteins are the major substrates for degradation, the autophagy-lysosomal pathway (ALP) is the dedicated machinery for the degradation of large-sized intracellular contents. Hence, defective autophagy has been associated with diverse human diseases, correlating with the failure to degrade cytotoxic aggregates and disruptions to cellular homeostasis. The acceleration or restoration of ALP therefore potentially provides strategies for therapeutic development.

[0006] There are two maj or limitations within the available strategies to regulate the degradation of impaired protein aggregates and organelles. First, small-molecule drugs that modulate autophagy are not specific towards dysfunctional targets. These autophagy modulators (e.g., Torin-1) affect the overall level of autophagy and have off-target concerns. Second, a majority of techniques for the targeted degradation of intracellular contents are introduced through the UPS. The targets of such methods are mainly proteins (e.g., PROTACs). as the UPS cannot efficiently process complex and large-sized contents. Therefore, a technological gap remains in the targeted degradation of large-sized substrates. In recent years, several targeted degradation techniques through the ALP have emerged, yet a universal strategy to the targeted degradation of protein aggregates and organelles is not available in mammalian cells.

[0007] The disclosure provided here provides solutions to the problems existing with previous attempts to target and degrade large-sized substrates and potentially offers improved methods for disease treatment related to protein aggregation and organelle dysfunction.SUMMARY

[0008] This section provides a general summary of the disclosure, and is not comprehensive of its full scope or all of its features.

[0009] The present disclosure provides a synthetic autophagy receptor comprising a first binding domain that specifically binds to an autophagosome, and a second binding domain that specifically binds to a target moiety, wherein the binding of the first binding domain to the autophagosome can result in the degradation of the target moiety bound to the second binding domain.

[0010] In some embodiments, the first binding domain is selected from the group consisting of natural ligands or a fragment, derivative, or small molecule mimetic thereof, antibodies and antigen binding fragments thereof.

[0011] In some embodiments, the first binding domain specifically binds to a protein selected from the group LC3A, LC3B, LC3C, GAB ARAP, GABARAOL1, and GABARAPL2 on the autophagosome.

[0012] In some embodiments, the first binding domain comprises an LC3 -interacting region (LIR).

[0013] In some embodiments, the LIR is derived from a protein selected from the group ATG13, FYCO1, NBR1, Nix / Bmp3L, p62, TP53INP1, TP53INP2 / DOR, ULK1, ULK2,Optineurin, FUNDCI, TBC1D5 LIR1, TBC1D5 LIR2. c-Cbl, P-catenin, Dvl2, MAP15K, Bnip3, ATG4B. Taxlbpl / T6BP, Stbdl , NDP52, Calreticulin. Clathrin HC. FIP200 and TBC1D25. In some embodiments, the LIR is selected from p62, TP53INP2, FYCO1, and TBC1D25. In some embodiments, the LIR is from p62. In some embodiments, the LIR is from TP53INP2

[0014] In some embodiments, the second binding domain comprises one or more antibodies.

[0015] In some embodiments, the target moiety comprises a protein, protein aggregate, protein fragment, organelle, or membraneless organelle. In some embodiments, the target moiety comprises a protein aggregate. In some embodiments, the protein aggregate comprises Ap, FUS, a-synuclein. tau, TDP-43. and / or huntingtin. In some embodiments, the target moiety comprises an organelle. In some embodiments, the organelle is selected from the group consisting of mitochondria, peroxisome, Golgi, and endoplasmic reticulum. In some embodiments, the organelle is decorated with SNAP-tag based protein constructs.

[0016] Also provided herein is a nucleic acid that encodes the synthetic autophagy receptor of the present disclosure.

[0017] In some embodiments, the nucleic acid is operably connected to a promoter.

[0018] Also provided herein is an engineered cell capable of protein expression comprising the nucleic acid of the present disclosure.

[0019] Also provided herein is a method for making a synthetic autophagy receptor, the method comprising providing a cell capable of protein synthesis, comprising the nucleic acid of present disclosure; and inducing expression of the synthetic autophagy receptor.

[0020] Also provided herein is a vector, comprising the nucleic acid of the present disclosure.

[0021] In some embodiments, the vector further comprises a promoter, wherein the promoter is operably linked to the nucleic acid.

[0022] Also provided herein is a pharmaceutical composition, comprising the synthetic autophagy receptor, the nucleic acid, the vector, the engineered cell, of the present disclosure, and a pharmaceutically acceptable excipient.

[0023] Also provided herein is a method of treating a disorder in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the synthetic autophagy receptor, the nucleic acid, the vector, the engineered cell, or the pharmaceutical composition of the present disclosure.

[0024] In some embodiments, the disorder comprises a neurological disorder. In some embodiments, the neurological disorder comprises Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0025] In some embodiments, the disorder comprises an infection by a pathogenic microorganism.

[0026] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGs. 1A-1H show the development of AceTAC degraders for targeted HTT-103Q degradation through protein engineering. FIG. 1A shows schematic illustrations of the first generation of AceTAC degraders (p62FL-An, where n represents the number of antibody units) and the workflow of the HiBiT luminescence assay for the quantification of targeted protein degradation. FIG. IB shows targeted degradation of HTT-103Q by the first- generation AceTAC degraders. FIG. 1C is a schematic illustration of the first generation AceTAC degrader (p62FL-A3) highlighting the functional domains / motifs of full-length p62. FIGs. ID- IE show targeted degradation of HTT-103Q by the deletion mutants of p62FL-A3 degraders (FIG. ID) and the p62FL-A3 constructs with LIR motifs derived from different LC3-binding proteins (FIG. IE). FIG. IF shows schematic illustrations of the second (TmAn) and third generation (AN-TmAn) of AceTAC degraders. For both TmAn and AN-TmAn, m represents the number of TP53INP2-LIR motifs and n represents the number of antibody units. FIGs. 1G-1H shows targeted degradation of HTT-103Q by the TmAn degraders (FIG. 1G) and the AN-TmAn degraders (FIG. 1H). Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significance.

[0028] FIG. 2 shows western blots of the AceTAC degrader expression in U2OS cells.

[0029] FIG. 3 shows targeted degradation of HTT-103Q by the deletion mutants of the T3A3 degrader. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. ****, p < 0.0001.

[0030] FIG. 4A shows relative mRNA level of ALFA-tagged HTT-103Q in U2OS cells after co-transfecting the HTT-103Q-encoding plasmid and the degrader-encoding / EVplasmid. The corresponding targeted HTT-103Q degradation profile was shown in FIG. 4B. FIG. 4C shows the relative mRNA level of ALFA-tagged HTT-103Q in U2OS cells after co-transfecting the HTT-103Q-encoding plasmid and the degrader-encoding / EV plasmid. The corresponding targeted HTT-103Q degradation profile was shown in (FIG. 4D). In the presence of autophagy-inducing plasmids, the mRNA level of HTT-103Q was slightly (-40%) reduced compared to mRNA in the cells with an empty vector as the second plasmid. However, the levels ofHTT-103Q degradation were strikingly lower when AceTAC degraders were transfected, supporting our proposal that AceTAC-induced targeted degradation occurred at the protein level. Similarly, for ALFA-tagged a-synuclein, cotransfection with p62FL-A3 resulted in the lowest level of a-synuclein mRNA, while the a- synuclein expression was not affected. Altogether, this dataset demonstrated that the cotransfection method did not confound our assessment of targeted protein degradation. Error bars represent standard deviations ofN = 3 (a,c) orN = 4 (b,d). Statistical analyses are performed using two-tailed Student’s t test to compare with the group that shows the least mRNA level. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; ns, no significance.

[0031] FIG. 5 shows targeted degradation of HTT-103Q could be modulated bydoxycycline (Dox)-inducible expression of AceTAC degraders. Briefly, the HTT-103Q- encoding plasmid and Dox-inducible degrader-encoding plasmid (or EV for the control group) were co-transfected into U2OS cells for 24 hours in the presence of varied concentrations of Dox. The transfected cells were further incubated for 6 hours in fresh complete growth medium that contained the corresponding Dox concentration. Error bars represent standard deviations of N = 3. Statistical analyses are performed using two-tailed Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significance.

[0032] FIGs. 6A-6E show targeted degradation of various proteins and protein aggregates by representative AceTAC degraders. HiBiT luminescence assay to quantify the degradation of TDP-43-Q331K mutant (FIG. 6A), FUS-P525L mutant (FIG. 6B), wild-ty pe a-Synuclein (FIG. 6C), Tau (FIG. 6D), and Tau-P301L mutant (FIG. 6E) by representative AceTAC degraders. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. **, p < 0.01; ***, p < 0.001 ; ****, p < 0.0001; ns, no significance. FIG. 6F shows western blots of protein targets in the presence of representative AceTAC degraders. Western blot experiments were independent of HiBiT luminescence assays.

[0033] FIGs. 7A-7E show HiBiT luminescence assay to quantify the degradation of TDP- 43-Q331K mutant (FIG. 7A), FUS-P525L mutant (FIG. 7B), wild-type a-synuclein (FIG. 7C), Tau (FIG. 7D), and Tau-P301L mutant (FIG. 7E) by the AN-TmAn degraders. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two- tailed Student’s t test. ***, p < 0.001; ****, p < 0.0001.

[0034] FIGs. 8A-8E show western blots of protein targets (ALFA-tagged) and degraders (HA-tagged) after 24-hour plasmid transfection in U2OS cells.

[0035] FIGs. 9A-9E show AceTAC degradation system induced tunable targeted degradation of mitochondria. FIG. 9A is a schematic illustration of the AceTAC system for targeted mitochondrial degradation. FIG. 9B shows representative images for the cellular localization of BFP-T3A3 degrader, MitoAnchor, and MitoTracker Deep Red (MitoTracker- DR) in live U2OS cells. Three representative cells were labeled: Cell# 1 , co-presence of BFP-T3A3 degrader and the MitoAnchor led to the aggregation of mitochondria; Cell#2, the MitoAnchor did not induce the mitochondrial aggregation in the absence of BFP-T3A3 degrader; Cell#3, a control cell showed regular tubular mitochondrial network when neither BFP-T3A3 nor MitoAnchor were transfected. Scale bar, 20 pm. FIG. 9C shows flow cytometry dot plot analyses (left) for the BFP-T3A3 degrader intensify and the MitoTracker- DR intensify in U2OS cells. The top left quadrant of the BFP-T3A3(-) population (QI) and the top right quadrant of the BFP-T3A3(+) population (Q2) were plotted for their corresponding histogram analyses for MitoTracker-DR in-tensity (right). FIG. 9D shows the effect of MitoAnchor expression on the reduction of MitoTracker-DR intensify from the BFP-T3A30 population (QI) to the BFP-T3A3(+) population (Q2). Error bars represent standard deviations of N = 3. FIG. 9E shows the effect of different BFP-T / MA / 7 degraders on the reduction of MitoTracker-DR intensify from the BFP(-) to the BFP(+) population. Error bars represent standard deviations of N = 3. Statistical analyses are performed using two- tailed Student’s t test. *, p < 0.05; **,p < 0.01; ***, < 0.001; **** , p < 0.0001; ns, no significance.

[0036] FIG. 10 shows western blots of the BFP-TmAn degrader expression in U2OS cells.

[0037] FIG. 11 shows representative images for the cellular localization of the BFP- T1 Al, MitoAnchor (stained by SNAP-Cell Oregon Green), and MitoTracker Deep Red staining in U2OS cells. Scale bar, 10 pm.

[0038] FIG. 12 shows flow cytometry histogram analyses for the expression of MitoAnchor-deGFP under different promoters in U2OS cells. The analysis was conductedafter transfecting the MitoAnchor-deGFP-encoding plasmids in U2OS cells for 24 hours. deGFP is a destabilized version of GFP with a 2-hour fluorescence half-life (Li, X.; Zhao. X.; Fang, Y.; Jiang, X.; Duong, T.; Fan, C.; Huang, C.-C.; Kain, S. R. Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter. J. Biol. Chem. 1998, 273. 34970-34975). Data in the table represent standard deviations of N = 4.

[0039] FIGs. 13A-13C show cell viability (alamarBlue) assay after the co-transfection of degrader-encoding and membrane-anchor-encoding plasmids in U2OS cells for 24 hours. Cells were highly viable in the presence of both the membrane anchor and the degraders / EV. EV, empty vector. Error bars represent standard deviations of N = 5. Statistical analyses are performed using two-tailed Student’s t test. *, p < 0.05; ns, no significance.

[0040] FIGs. 14A shows the effect of different BFP-based control constructs on the MitoTracker-DR intensity in U2OS cells. Error bars represent standard deviations of N = 4. FIGs. 14B-14C show the effect of different mPlum-based control constructs on GFP- labelled peroxisomes (FIG. 14B) and GFP-labelled ER (FIG. 14C) in U2OS cells. The quantification was conducted by flow cytometry analysis. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. *. p < 0.05; **, p < 0.01 ; ***, p < 0.001; ns, no significance.

[0041] FIG. 15 show s representative images for the cellular localization of the BFP-based constructs, MitoAnchor (stained by SNAP -Cell Oregon Green), and MitoTracker Deep Red staining in U2OS cells. Scale bar. 10 pm.

[0042] FIGs. 16A-16D show AceTAC degradation system induced targeted degradation of peroxisomes and endoplasmic reticulum (ER). FIG. 16A is a schematic illustration of the AceTAC degradation system design for GFP-labelled organelles. FIGs. 16B-16C show targeted degradation of GFP-labelled peroxisomes (FIG. 16B) and GFP-labelled ER (FIG. 16C) by the mPlum-T / wA / 7 degraders in U2OS cells measured by flow cytometry. Error bars represent standard deviations of N= 3. *,p < 0.05; **, / ? < 0.01; ***, p < 0.001; ****, > < 0.0001. FIG. 16D shows representative images for the cellular localization of mPlum-TlAl degrader, GFP-labelled organelles (including peroxisome, ER, and Golgi apparatus), and the corresponding Membrane-anchor in live U2OS cells. The colocalized spots of these three channels were shown as white puncta in the merged channel. The merged channel also includes the nuclear staining by Hoechst 33342 (shown in blue). Scale bar, 10 pm.

[0043] FIGs. 17A-17F show flow' cytometry dot plot analyses (FIG. 17A, FIG. 17C, FIG. 17E) for the mPlum-Tl Al degrader intensity and the organelle-GFP intensity in U2OS cells. The top left quadrant of the mPlum-TlAl(-) population (QI) and the top rightquadrant of the mPlum-TlAl(+) population (Q2) were plotted for their corresponding histogram analyses of the organelle-GFP intensity (FIG. 17B, FIG. 17D, FIG. 17F).

[0044] FIG. 18 shows the effect of mPlum-TmAn degraders on GFP-labelled Golgi apparatus in U2OS cells. The quantification was conducted by flow cytometry analysis. Error bars represent standard deviations of N = 3. Statistical analyses are performed using two-tailed Student’s t test. *, p < 0.05; ns, no significance.

[0045] FIG. 19 shows representative images for the cellular localization of the GFP- labelled peroxisome, mPlum-based constructs, and Membrane anchor (for peroxisomes; Stained by SNAP-Cell 647-SiR) in U2OS cells. The merged channel also includes the nuclear staining by Hoechst 33342 (shown in blue). Scale bar, 10 pm.

[0046] FIG. 20 shows representative images for the cellular localization of the GFP- labelled endoplasmic reticulum (ER), mPlum based constructs, and Membrane anchor (for ER; Stained by SNAP-Cell 647-SiR) in U2OS cells. The merged channel also includes the nuclear staining by Hoechst 33342 (shown in blue). Scale bar, 10 pm.

[0047] FIG. 21 shows representative images for the cellular localization of the GFP- labelled Golgi apparatus, mPlum-based constructs, and Membrane anchor (for Golgi apparatus; Stained by SNAP-Cell 647-SiR) in U2OS cells. The merged channel also includes the nuclear staining by Hoechst 33342 (shown in blue). Scale bar, 10 pm.

[0048] FIGs. 22A-22E show the targeted degradation induced by AceTAC degraders utilized the autophagy process. FIG. 22A shows representative images for the cellular localization of the AceTAC degraders (T3A3 and AN-T3A3), HTT-103Q target, endogenous p62, and endogenous LC3B in U2OS cells. The plasmids encoding the AceTAC degraders and HTT-103Q w ere transfected to U2OS cells for 24 hours. Scale bar, 10 pm. FIG. 22B shows Pearson correlation coefficient (r) between HTT-103Q and LC3B from the IF image analysis of 30 representative cells in each group. Statistical analyses are performed using two-tailed Student’s t test. **** p < 0.0001. FIG. 22C shows co-immunoprecipitation (co-IP) of HTT-103Q from U2OS cells after transfection of plasmids that encode HTT-103Q and AceTAC degraders. HTT-103Q was immunoprecipitated via the ALFA-tag; HTT- 103Q-bound protein complexes were then captured from the lysates and blotted for the ALFA-tag (HTT-103Q) and HA-tag (AceTac degrader). FIG. 22D shows a HiBiT luminescence assay to evaluate the effect of ATG7-knockdown on the targeted HTT-103Q degradation by representative AceTAC degraders in U2OS cells. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. *, p < 0.05; **, ? < 0.01; ns, no significance. FIG. 22E shows representative images forthe cellular localization of BFP-T3A3 degrader, MitoAnchor, LC3B, and MitoTracker-DR in live U2OS cells. Colocalization occurs at a large cytoplasmic aggregate. Scale bar, 20 pm.

[0049] FIG. 23 shows representative images for the cellular localization of representative AceTAC degraders (T3A3 and AN-T3A3), HTT-103Q target, endogenous p62, and endogenous LC3B in U2OS cells. Each channel in Figure 5a was provided in greyscale and the merged channel was color-coded. Scale bar, 10 pm.

[0050] FIG. 24 shows representative images for the cellular localization of the p62- overexpression (HA-tagged), HTT-103Q target, endogenous p62, and LC3B in U2OS cells. Scale bar, 10 pm.

[0051] FIG. 25 shows representative images for the cellular localization of the control constructs, HTT-103Q target, endogenous p62. and LC3B in U2OS cells. Scale bar. 10 pm.

[0052] FIG. 26 shows representative images for the cellular localization of HA-tagged constructs, HTT-103Q target, endogenous p62, and LC3B in U2OS cells, comparing the effect of removing the LIR motif in AceTAC degraders. Scale bar, 10 pm.

[0053] FIGs. 27A-27D show the workflow of the co-immunoprecipitation (co-IP) (FIG. 27A, FIG. 27C) and western blots for cells after plasmid transfection (FIG. 27B, FIG. 27D). Western blots for degraders (HA-tagged), HTT-103Q target (ALFA-tagged), and vinculin in U2OS cells were analyzed after 24-h co-transfection of plasmids that encode the degrader and the target. For each group, input proteins in the lysates (IN) before the co-IP of HTT-103Q (or HA-tagged degraders) were compared with the unbound proteins in the lysates (UNB). Control, U2OS cells with only the target-encoding plasmid transfected.

[0054] FIGs. 28A-28B show dose selection for CCCP-treatment to induce mitophagy in U2OS cells. FIG. 28A shows inhibition of the cellular metabolic activity' by the 24-hr treatment of CCCP (carbonyl cyanide m-chlorophenyl hydrazone) in U2OS cells. The fitted curve (red line) showed an IC50 value at 31.34 pM. Alamar Blue assay was used for the readout of resorufin fluorescence. Error bars represent standard deviations of N = 6. FIG. 28B shows representative images for the mitochondrial morphology7in live U2OS cells after 24-hr CCCP treatment. From the comparison, the dose for CCCP-treatment was chosen to be 25 pM to induce mitophagy. Scale bar, 10 pm.

[0055] FIG. 29A shows representative western blots for selected proteins to evaluate mitophagy in U2OS cells. The CCCP dose was 25 pM for 24-hr treatment. MitoAnchor contains a C-terminal ALFA-tag and BFP-T1A1 AceTAC degrader contains a C-terminal HA-tag. Vinculin was used as the loading control. FIG. 29B shows quantification of western blots for the selected proteins in U2OS cells. Control group, U2OS cells without drugtreatment or plasmid transfection. Combo group, co-presence of the MitoAnchor and the BFP-T1 Al degrader in U2OS cells. Error bars represent standard deviations of N = 3. Statistical analyses are performed using two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.

[0056] FIGs. 30A-30B show Western blot analysis to validate the successful knockdown of ATG7 in U2OS cells. FIG. 30A shows representative western blotsfor ATG7 and vinculin in U2OS cells after transfecting siRNA against ATG7 expression for 48 hours. FIG. 30B shows quantification of ATG7 expression level after ATG7-knockdown by RNA interference. Error bars represent standard deviations of N = 3. Statistical analyses are performed using two-tailed Student’s t test. ***, p < 0.001.

[0057] FIGs. 31A-31B show Western blot analysis to validate the successful knockdown of ATG5 in U2OS cells. Representative western blots for ATG5 and GAPDH in U2OS cells after transfecting siRNA against ATG5 expression for 48 hours (FIG. 31A). Quantification of ATG5 expression level after ATG5-knockdown by RNA interference (FIG. 31B). Error bars represent standard deviations of N = 4. Statistical analyses are performed using two- tailed Student’s t test. ***, p < 0.001. FIG. 31C shows a HiBiT luminescence assay to evaluate the effect of ATG5-knockdown on the targeted HTT-103Q degradation by representative AceTAC degraders in U2OS cells. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student's t test. *, p < 0.05; ns, no significance.

[0058] FIGs. 32A-32C show HiBiT luminescence assay to evaluate the effect of 250 nM Torinl (FIG. 32A), serum starv ation (FIG. 32B), and 100 nM Bafilomycin-Al (FIG. 32C) on the targeted HTT-103Q degradation by representative AceTAC degraders in U2OS cells. Error bars represent standard deviations of N = 4. The autophagy-inducing (Torin-1 treatment or serum-starvation) and -inhibition (Bafilomycin-Al treatment) conditions were applied after the 24-hour co-transfection of the target-encoding and degrader-encoding plasmids, possibly resulting in the subtle effects on the targeted protein degradation efficiency due to the limited treatment time. However, the result may be further complicated if these autophagy-modulating conditions were applied before plasmid transfection, e.g., possibly affecting the transfection efficiency. Statistical analyses are performed using two- tailed Student’s t test. *, p < 0.05; **, p < 0.01; ns, no significance.

[0059] FIGs. 33A-33F show The LIR motif of TP53INP2 in the AceTAC degrader modulated the autophagic flux, enhancing the targeted degradation efficiency of proteins that are autophagy substrates. FIG. 33A shows representative western blots for p62, LC3B,ALFA-tagged target (a-synuclein), HA-tagged construct, and vinculin in U2OS after transfecting the construct-encoding plasmid for 24 hours. ACtrl, control nanobody against RNase A. AN-T1 ACtrl has a TP53INP2 LIR motif linked with the ACtrl. An, nanobody against the ALFA-tag; n represets the number of antibody repeats. FIGs. 33B-33D show quantification of western blots for the p62 expression level (FIG. 33B), the LC3B-II / I ratio (FIG. 33C). and ALFA-tagged a-synuclein (FIG. 33D) in U2OS cells. The increase of p62 expression and LC3-II / I ratio along with the improved a-synuclein degradation after the addition of TP531NP2 LIR motif (AN-T1 ACtrl vs. ACtrl; AN-T1 Al vs. Al) indicates the enhancement of autophagic flux. Error bars represent standard deviations of N = 2. FIGs. 33E-33F show HiBiT luminescence assay to evaluate the effect of TP53INP2 LIR motif on the selected AceTAC degraders towards the degradation of HTT-103Q (FIG. 33E) and survivin (FIG. 33F) degradation in U2OS cells. Error bars represent standard deviations of N = 4. For a-synuclein and HTT-103Q, either AN-TlACtrl or the Al group have caused the protein degradation. The effect from AN-T1 ACtrl can possibly be attributed to the enhanced autophagic flux. The binding between ALFA-tag nanobody (Al) and its protein targets induced puncta formation of the target and colocalization with endogenous p62 and LC3B, triggering the autophagy machinery. For both a-synuclein and HTT-103Q, the combination between the TP53INP2-LIR motif and the ALFA-tag nanobody led to an increased protein degradation efficiency than each component alone (AN-T1A1 vs. AN-T1 ACtrl or Al). For survivin, the presence of either ACtrl or AN-T1 ACtrl minimally affected the survivin level when compared with the EV group. In contrast, the survivin level was significantly increased in the presence of p62-O / E, Al, or AN-T1A1. It has been shown that the accumulation of p62 inhibits the degradation of ubiquitinated proteins destined for proteasomal clearance by delaying their delivery to the proteasome (Korolchuk, V. I.; Mansilla, A.; Menzies, F. M.; Rubinsztein, D. C. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol. Cell 2009, 33, 517-527). As survivin is a proteasomal substrate, (Vong, Q. P.; Cao, K.; Li, H. Y.; Iglesias, P. A.; Zheng, Y. Chromosome alignment and segregation regulated by ubiquitination of survivin. Science 2005, 310, 1499-1504) it is therefore reasonable that the increased p62 level in either the p62-O / E or AN-T1 Al group may have caused the excess accumulation of survivin. The tight binding between Al and ALFA-tagged survivin may have contributed the accumulation of survivin. However, these hypotheses shall be further validated. Overall, this dataset indicates that the AceTAC degraders are more effective against proteins that are autophagy substrates rather than proteasomal substrates.

[0060] FIG. 34 shows a HiBiT luminescence assay to evaluate the effect of 200 nM carfilzomib on the targeted HTT-103Q degradation by representative AceTAC degraders in U2OS cells. The fold change describes the increase of the HiBiT luminescence value after carfilzomib-treatment. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test, ns, no significance.

[0061] FIGs. 35A-35C shows generation of p62-knockout (p62-KO) U2OS cells. Flow cytometry histograms of U2OS cells transfected with p62-KO plasmids (containing EGFP- encoding sequence) are shown in FIG. 35A. Western blots for p47 and P-actin in U2OS single cell clones are shown in FIG. 35B. PCR amplification of the genomic DNA in wildtype (wt) U2OS cells and selected U2OS clones after p62-KO-plasmid transfection and cell sorting is shown in FIG. 35C. FIG. 35D shows a HiBiT luminescence assay to evaluate the effect of p62-KOon the targeted HTT-103Q degradation by representative AceTAC degraders in U2OS cells. Error bars represent standard deviations of N = 4. Statistical analyses are performed using two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, no significance.

[0062] FIG. 36 shows the workflow of the AceTAC degraders for targeted degradation of protein aggregates and organelles.

[0063] In the following detailed description, reference is made to the Figures, which form a part hereof. In the Figures, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.DETAILED DESCRIPTION OF THE DISCLOSURE

[0064] In general, the present disclosure relates, inter alia, to compositions and methods for targeted degradation of protein aggregates and organelle via autophagy. During autophagy, the selection of autophagosomal substrates is aided by autophagy receptors, a family of proteins that simultaneously interact with both the substrates and autophagosomal membranes. For example, sequestosome-1 (SQSTMl / p62) is a major mammalian autophagyreceptor. The ubiquitin-associated (UBA) domain of p62 interacts with ubiquitinylated (Ub-) substrates to form aggregates during selective autophagy. These aggregates are driven into the forming autophagosome through the interaction between the LC 3 -interacting region (LIR) of p62 and the LC3 / GABARAP proteins on the autophagosomal membranes. Based on the protein-protein interactions of p62 during autophagy, the inventors have developed synthetic autophagy receptors, termed autophagy receptor-inspired targeting chimeras (AceTACs). The design of the AceTAC was developed through the protein engineering of three generations of antibody (Ab)-fusion degraders, resulting in an efficient method to transport the targeted protein into the autophagy process for eventual degradation. Furthermore, the AceTAC degradation technology was customized for targeted degradation of specific organelles, including mitochondria, peroxisomes, and the endoplasmic reticulum (ER), demonstrating its broad potential as a targeted degradation platform. The ability of the AceTAC degradation technology to result in targeted degradation, as described in the Examples below, was both surprising and unexpected given the large size of organelles as compared to the initially tested monovalent construct. Therefore, it is likely degradation could be enhanced even further through the engineering of multiple binding domains to the target organelle.

[0065] Although various features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein byreference for any purpose. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0066] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It wi 11 be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.DEFINITION

[0067] The singular form “a’', "an", and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0068] The terms “administration” and “administering”, as used interchangeably herein, refer to the delivery of a composition or formulation by an administration route including, but not limited to. intravenous, intra-arterial, intracerebral, intrathecal, intramuscular, intraperitoneal, subcutaneous, intramuscular, and combinations thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0069] The terms “host cell” and “recombinant cell” are used interchangeably herein. It is understood that such terms, as well as “cell culture”, “cell line”, refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell or cell line, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell or cell line.

[0070] The term “operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.

[0071] The term “heterologous”, refers to nucleic acid sequences or amino acid sequences operably linked or otherwise j oined to one another in a nucleic acid construct or chimeric polypeptide that are not operably linked or are not contiguous to each other in nature.

[0072] The term “percent identity ,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection. See. e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. This definition also refers to, or may be applied to. the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity typically is calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof

[0073] The term ‘‘treatment” used in reference to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition being treated. Treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention (i.e., reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression), and (ii) inhibition (i.e., arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease).

[0074] As used herein, and unless otherwise specified, a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contributeto the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a "‘therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g.. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd. The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0075] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” can be a patient under the care of a physician. Thus, the subject can be a human patient or an individual w ho has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e g., rodents, e.g., mice, and non- mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0076] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed w ithin the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0077] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than.” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1. 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0078] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and even' such sub-combination was individually and explicitly disclosed herein.

[0079] Although features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference for any purpose. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0080] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0081] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly- understood that, although a number of information sources, including scientific j oumal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0082] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill inthe art upon review of this disclosure, and are to be included within the spirit and purview of this application.AUTOPHAGY

[0083] Autophagy refers to the catabolic process involving the degradation of a cell's own components; such as, long lived proteins, protein aggregates, cellular organelles, cell membranes, organelle membranes, and other cellular components. The mechanism of autophagy may include: (i) the formation of an autophagosome, which is a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm, (ii) the fusion of the resultant vesicle with a lysosome and the subsequent degradation of the vesicle contents. For example, the term autophagy can refer to one of the mechanisms by which a starving cell re-allocates nutrients from unnecessary processes to more essential processes. Also, for example, autophagy can inhibit the progression of some diseases and play a protective role against infection by intracellular pathogens.COMPOSITIONS OF THE DISCLOSURE

[0084] The present disclosure provides, among others, synthetic autophagy receptors comprising a first binding domain and a second binding domain. The first binding domain can specifically bind to an autophagosome. Further, the second binding domain can specifically bind to a target moiety. In some embodiments, the binding of the first binding domain to the autophagosome results in the degradation of a target moiety bound to the second binding domain. In some non-limiting exemplary' embodiments, the present disclosure demonstrates the development of a new targeted degradation platform technology, termed autophagy receptor-inspired targeting chimeras (AceTACs). which includes fully recombinant synthetic autophagy receptors that utilize the autophagy process to target various therapeutically relevant proteins and / or organelles for degradation (FIG. 36).

[0085] The disclosure also provides, among others, nucleic acids that encode the synthetic autophagy receptors, cells comprising the nucleic acid, and pharmaceutical compositions comprising the synthetic autophagy' receptors. The disclosure also provides methods of treatment using synthetic autophagy' receptors, nucleic acids encoding synthetic autophagy' receptors or pharmaceutical compositions comprising the synthetic autophagy receptors. The disclosure also provides compositions and methods useful for producing such agents, nucleicacids encoding same, host cells genetically modified with the nucleic acids, as well as methods for the treatment of diseases.

[0086] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.Synthetic Autophagy ReceptorsFirst Binding Domain

[0087] The synthetic autophagy receptors provided herein compnse a first binding domain and a second binding domain. The first binding domain can specifically bind to an autophagosome.

[0088] As used herein, an autophagosome refers to a double membrane vesicle that encloses cellular constituents and fuses with lysosomes.

[0089] The first binding domain of the synthetic autophagy receptors provided herein can be a natural ligand to a protein expressed on an autophagosomal membrane, or an isofonn or a derivative capable of binding thereof. A functional derivative of a natural ligand can be any agent that possesses the binding activity the natural ligand. For example, in some embodiments, the first binding domain of the bispecific binding agents can be a natural autophagy receptor, or fragment thereof, that binds to a protein on the autophagosomal membrane. In other embodiments, the first binding domain of the synthetic autophagyreceptor can be a binding agent (e.g., an antibody or a fragment thereof, a peptide, or a small molecule) that binds to the autophagosomal membrane.

[0090] Autophagy' receptors are known in the art and described in, for example, Zaffagnini et al., “Mechanisms of Selective Autophagy','’ J. Mol. Bio. 428: 1714-1724 (2016). As described above, autophagy receptors are a family of proteins that simultaneously interact with both the substrates and autophagosomal membranes. An exemplary autophagy receptor is sequestosome-1 (SQSTMl / p62), which is a major mammalian autophagy receptor. Theubiquitin-associated (UBA) domain of p62 interacts with ubiquitinylated (Ub-) substrates to form aggregates during selective autophagy’. These aggregates are driven into the forming autophagosome through the interaction between the LC3-interacting region (LIR) of p62 and the LC3 / GABARAP proteins on the autophagosomal membranes.

[0091] Therefore, in some embodiments, the first binding domain of the synthetic autophagy receptor described herein is from p62. As described herein, previous reports have shown the participation of five p62 domains / motifs during selective autophagy. These domains include, the LIR motif, the UBA domain, the PB1 domain, the ZZ domain, and the NES motif. In some embodiments, the first binding domain comprises the full length p62 protein. In some embodiments, the first binding domain does not include the PB 1 domain of p62. In some embodiments, the first binding domain does not include the UBA domain of p62. In some embodiments, the first binding domain does not include the ZZ domain of p62. In some embodiments, the first binding domain does not include the NES domain of p62. In some embodiments, the first binding domain does not include the LIR domain of p62. In some embodiments, the first binding domain comprises only the LIR domain of p62.

[0092] In some embodiments, the first binding domain binds to LC3 on the autophagosome. LC3 refers to microtubule-associated protein light chain 3 (LC3). LC3 is positioned on the pre-autophagosome and on the surface of the autophagosome membrane, and is widely used as an autophagosome membrane marker. LC3 includes all forms of LC3 including LC3 I or LC3 II. The term ”LC3 I” refers to cytosolic LC3, whereas the term “LC3 II” refers to membrane bound LC3. LC3 II is present both inside and outside autophagosomes. In some embodiments, the LC3 is LC3A, LC3B, LC3C, GABARAP, GABARAOL1, or GABARAPL2. Any binding domain capable to binding to LC3 is contemplated for use as a first binding domain of the synthetic autophagy receptor described herein. In some embodiments, the first binding domain of the synthetic autophagy receptor can be a natural autophagy receptor, or fragment thereof, that binds to LC3. In other embodiments, the first binding domain of the synthetic autophagy7receptor can be a binding agent (e.g., an antibody or a fragment thereof, a peptide, or a small molecule) that binds to LC3.

[0093] Binding to LC3 is mediated through an LC3 -interacting region (LIR), also known as LC3 recognition sequence (LRS) or Atg8-interacting motif (AIM). Thus, the presence of a LIR appears can be an indicator of LC3-interacting proteins. In some embodiments, the first binding domain comprises an LIR. For the purposes of the first binding domain of the synthetic autophagy receptor, the LIR does need to be from a natural autophagy receptor. In some embodiments, the LIR is from a natural autophagy receptor.

[0094] The LIR corresponds to the shortest sequence required for the interaction with an LC3 protein. Based on multiple alignments of LIR sequences from proteins, a consensus LIR motif of (ADEFGLPRSK)(DEGMSTV)(WFY)(DEILQTV)(ADEFHIKLMPSTV)(ILV) has been described (Jacomin et al., “iLIR Database: A Web Resource for LIR Motif-Containing Proteins in Eukaryotes,'’ Autophagy' 12(10): 1945-1953 (2016).

[0095] Numerous proteins are known to contain LIR motifs ((Jacomin et al., “iLIR Database: A Web Resource for LIR Motif-Containing Proteins in Eukaryotes,” Autophagy 12(10): 1945-1953 (2016)). In some embodiments, the LIR motif of the synthetic autophagy7receptor described herein is derived from ATG13, FYCO1, NBR1, Nix / Bnip3L, p62, TP53INP1, TP53INP2 I DOR, ULK1, ULK2, Optineurin, FUNDCI, TBC1D5 LIR1, TBC1D5 LIR2, c-Cbl, P-catenm, Dvl2, MAP15K, Bmp3, ATG4B, Taxlbpl / T6BP, Stbdl, NDP52, Calreticulin, Clathrin HC, FIP200 and TBC1D25. In some embodiments, the LIR is from p62, TP53INP2, FYCO1, and TBC1D25. In some embodiments, the LIR is from p62. In some embodiments, the LIR is from TP53INP2.

[0096] In some embodiments, the first binding domain can be monovalent. In some embodiments, the first binding domain can be multivalent.Second Binding Domain

[0097] The synthetic autophagy7receptor provided herein further includes a second binding domain that can specifically bind to a target moiety. The second binding domain can be a natural ligand to a target moiety, or an isoform or a derivative capable of binding thereof. As described above, a functional derivative of a natural ligand can be any agent that possesses the binding activity the natural ligand. For example, in some embodiments, the second binding domain of the synthetic autophagy receptor can be a natural receptor, or fragment thereof, that binds to the target moiety. In other embodiments, the second binding domain of the synthetic autophagy7receptor can be a binding agent (e.g., an antibody or a fragment thereof, a peptide, or a small molecule) that binds to the target moiety.

[0098] In some embodiments, the first binding domain can be monovalent. In some embodiments, the first binding domain can be multivalent.

[0099] The term “antibody” can include intact antibodies and binding fragments thereof that specifically bind to a single antigen or that specifically bind to multiple antigens (e.g., multispecific antibodies such as a bispecific antibody, a trispecific antibody, etc.). Thus, anyreference to an antibody should be understood to refer to the antibody in intact form or a binding fragment unless the context requires otherwise.

[0100] The term “binding fragment,” which can be used interchangeably with “antigenbinding fragment,” can refer herein to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that specifically binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody. a Fab, a Fab’, a F(ab’)2, a F(ab)c, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a triabody, a tetrabody, a single-chain antibody molecule (scFv), an scFv dimer, a multispecific antibody, a camelized single domain antibody, a nanobody, a minibody, a domain antibody, a bivalent domain antibody, a IgNAR, a V-NAR, and a hcIgG. Binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunogl obulins .

[0101] Various target moi eties are contemplated for use in the second binding domain of the synthetic autophagy receptors described herein. In some embodiments, the target moiety can be a protein, protein aggregate, protein fragment, organelle, or membraneless organelle.

[0102] In some embodiments, the target protein of the synthetic autophagy7receptor provided herein can be protein aggregate. The term “protein aggregate” can refer to an accumulation of two or more misfolded proteins. A protein aggregate can be comprised of recombinant protein, naturally occurring protein, or a combination thereof. The term “protein”, as used herein, includes peptides, polypeptides, fusion proteins, naturally occurring proteins, and recombinant or artificially synthesized proteins, as well as analogs, fragments, derivatives or combinations thereof. As used herein, “recombinant protein” can refer to a protein that is encoded by a nucleic acid sequence that is not ty pically present in the wild-type genome of the cell expressing it. Methods of making and expressing recombinant protein are well known in the art. As used herein, “naturally occurring protein” can refer to a protein that is encoded by a nucleic acid sequence that is typically present in the wild-type genome of the cell expressing it. A protein aggregate may or may not be associated with a disease or disease pathology.

[0103] In one embodiment, a protein aggregate may be comprised of any protein with an aggregation-prone domain. The term “aggregation-prone domain” can refer to a region of the amino acid sequence of a protein that promotes the protein's aggregation. By way of example, the tau protein has either three or four repeat regions that constitute theaggregation-prone core of the protein, and the androgen receptor (AR) and huntingtin (htt) have expanded tracts of glutamines that contribute to formation of perinuclear and nuclear aggregates of these proteins. In some embodiments, an aggregation-prone domain is unique to a single protein. In other embodiments, an aggregation prone domain may be common to more than one protein. Aggregation-prone domains are well known in the art, or may be predicted through computational modeling.

[0104] In some embodiments, a protein aggregate may be comprised of a pathological protein. The term ‘'pathological protein”, as used herein, can refer to a protein that aggregates, whereby aggregation is closely linked to disease pathology. Pathological proteins are well-known in the art. A pathological protein can be a polyglutamine expansion protein or anon-poly glutamine expansion protein.

[0105] Polyglutamine expansion diseases are a class of neurodegenerative diseases associated with pathological aggregation of a protein containing expanded tracts of glutamines (e.g. a poly glutamine expansion protein). Pathological poly glutamine expansion proteins (and their related disorders) may include, but are not limited to, htt (Huntington's disease), androgen receptor (AR; spinobulbar muscular atrophy), ATN 1 (dentatorubropallidoluysian atrophy), ATXN1 (Spinocerebellar ataxia Type 1), ATXN2, (Spinocerebellar ataxia Type 2), ATXN3, (Spinocerebellar ataxia Type 3), CACNA1 A (Spinocerebellar ataxia Type 6), ATXN7 (Spinocerebellar ataxia Type 7), and TBP (Spinocerebellar ataxia Type 17).

[0106] Non-limiting examples of non-poly glutamine expansion proteins include tau, synuclein, superoxide dismutase (SOD1), PABPN1, amyloid beta peptide, serpin, transthyretin, TDP-43 (TARDBP), valosin containing peptide (VCP), hnRNPA2Bl and hnRNPAl and prion protein.

[0107] Tauopathies are class of neurodegenerative diseases associated with the pathological aggregation of tau protein into fibrillar tau aggregates. Exemplary disorders that have clinical signs or symptoms associated with tau aggregation include, but are not limited to, progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-dementia complex of Guam), tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), Frontotemporal lobar degeneration, Alzheimer's Disease, and frontotemporal dementia.

[0108] Exemplary diseases that have symptoms associated with SOD1 aggregation may include amyotrophic lateral sclerosis (Lou Gehrig's disease). Exemplary disorders that have symptoms associated with PABPN1 aggregation may include oculopharyngeal muscular dystrophy.

[0109] Exemplary diseases that have symptoms associated with synuclein aggregation may include Parkinson's disease. Alzheimer's disease, Lewy body disease and other neurodegenerative diseases.

[0110] Exemplary diseases that have symptoms associated with serpin aggregation (“serpinopathies”) may include alpha I -antitrypsin deficiency which may cause familial emphysema and liver cirrhosis, certain familial forms of thrombosis related to antithrombin deficiency, types 1 and 2 hereditary angioedema related to deficiency of Cl -inhibitor, and familial encephalopathy with neuroserpin inclusion bodies.

[0111] Exemplary diseases that have symptoms associated with transthyretin aggregation may include senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy.

[0112] Exemplary diseases that have symptoms associated with prion aggregation may include scrapie, bovine spongiform encephalopathy (mad cow disease), transmissible mink encephalopathy, chronic wasting disease, feline spongiform encephalopathy, exotic ungulate encephalopathy. Creutzfeldt-Jakob diseases, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and Kuru.

[0113] Exemplary diseases that have symptoms associated with TDP-43 aggregation may include FTLD-TDP and chronic traumatic encephalopathy.

[0114] Exemplary diseases that have symptoms associated with amyloid beta peptide aggregation may include Alzheimer's disease, Lewy body disease, cerebral amyloid angiopathy, inclusion body myositis and traumatic brain injur .

[0115] Exemplary diseases that are associated with VCP aggregation include Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD).

[0116] Exemplary diseases caused by hnRNPA2Bl and hnRNPAl include multisystem proteinopathy and ALS.

[0117] In some embodiments, a protein aggregate may be in a biological sample. Suitable biological samples include tissue samples or bodily fluids. The choice of the sample w ill depend, in part, upon the protein aggregate. For example, if a protein aggregate is typically found in the central nervous system or is associated with a neurodegenerative disease, nonlimiting examples of suitable tissue may include brain tissue, spinal cord tissue and centralnervous system (CNS) microvascular tissue, and non-limiting examples of suitable biological fluids include cerebral spinal fluid, interstitial fluid, blood, serum or plasma. Alternatively, if a protein aggregate is typically found in the periphery or is associated with a peripheral disease, non-limiting examples of suitable tissue may include blood, serum, plasma, saliva, sputum, lymph or urine. Tissue samples may be processed into a homogenate, a cell extract, a membranous fraction, or a protein extract. Biological fluids may be used “as is”, the cellular components may be isolated from the fluid, or a protein faction may be isolated from the fluid using standard techniques. The method of collecting a sample can and will vary7depending upon the nature of the sample. Methods for collecting tissue and biological fluid samples are well known in the art. Generally speaking, the method preferably maintains the integrity of the sample such that conformation of the protein aggregate can be accurately analyzed according to the invention. Further processing of a biological sample may be necessary after collection of the biological sample but before using a biological sample, as well as a fraction or components obtained therefrom, in a method of the invention. For example, a biological sample may be concentrated in order to provide a greater amount of the protein aggregate. or protein aggregate may be partially or completely purified from other components of a biological sample. Suitable methods to separate a protein aggregate from other components of a biological sample include, but are not limited to, chromatography, immunoprecipitation, affinity purification, and adsorption.

[0118] Biological samples may be obtained from any suitable subject. Typically, the subject is diagnosed with a disease associated with pathological protein aggregation, preferably of an amyloid protein, or a subject with clinical signs or symptoms of a disease associated with the pathological protein aggregation, preferably of an amyloid protein. Suitable subjects may include a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, a subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, a subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, a subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, a subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include nonhuman primates, large cats, wolves, and bears. In a preferred embodiment, a subject is human.

[0119] In one embodiment, a protein aggregate may be comprised of pathological protein associated with a neurodegenerative disease. In another specific embodiment, a protein aggregate may be compnsed of protein selected from the group consisting of prion protein, tau protein, alpha-sy nuclein protein, amyloid beta peptide, TDP- 43, and htt. In another specific embodiment, a protein aggregate may be an amyloid. In another specific embodiment, a protein aggregate may be comprised a protein selected from the group consisting of amyloid beta peptide. PrPsc, huntingtin, calcitonin, apolipoprotein Al, transthyretin, tau, and cystatin. In another specific embodiment, a protein aggregate may be comprised a protein selected from the group consisting of atrial natriuretic factor, apolipoprotein Al, serum amyloid, medin, prolactin, lysozyme, Beta 2 microglobulin, gelsolin, keratoepithelin, immunoglobulin light chain AL, and S-IBM.

[0120] In some embodiments, the target moiety of the synthetic autophagy receptor provided herein can be an organelle. Organelles are specialized structures within a cell that cany' out specific functions that are critical for its function and survival. As described in the Examples provided herein, the synthetic autophagy receptor of the present disclosure is able to target organelles for degradation via selective autophagy’. In some embodiments, the organelle is decorated with a SNAP tag based constructs as described in the Examples provided herein. Mechanisms of autophagy' of organelles is described in the art, for example, in Anding et al., “Cleaning House: Selective Autophagy of Organelles,’" Dev Cell 41(1): 10- 22 (2017) and Li et al., “Selective Autophagy of Intracellular Organelles: Recent Research Advances,” Theranostics 11(l):222-256 (2021). Exemplary organelles contemplated as target moieties of the present dislcosure include, without limitation, mitochondria, proteasome, peroxisome, Golgi, endoplasmic reticulum, lysosome, and nucleus.

[0121] In some embodiments, the target protein of the synthetic autophagy’ receptor provided herein can be a membraneless organelle. Membraneless organelles are known in the art and diescribed, for example, in Hirose et al., “A Guide to Membraneless Organelles and Their Various Roles in Gene Regulation,” Nature Reviews Molecular Cell Biology 24:288-304 (2023). Some non-limiting examples of membraneless organelles include nucleolus. Cajal bodies, nuclear speckles, paraspeckles, PML nuclear bodies, nuclear stress bodies, P bodies, and stress granules.

[0122] The synthetic autophagy' receptors of the present disclosure can generally take the form of a protein, glycoprotein, lipoprotein, phosphoprotein, and the like. Some synthetic autophagy receptors of the disclosure take the form of bispecific antibodies or antibody denvatives. In some embodiments, the first binding domain and the second binding domainof the synthetic autophagy receptor provided herein can each be independently selected from the group consisting of natural ligands or a fragment, derivative, or small molecule mimetic thereof antibodies, half antibodies, single-domain antibodies, nanobodies, Fabs, monospecific Fab2, Fc, scFv, minibodies, IgNAR, V-NAR, hdgG, VHH domain, camelid antibodies, and peptibodies. In some embodiments, the first binding domain and the second binding domain of the synthetic autophagy receptors provided herein together can form a bispecific antibody, a bispecific diabody. a bispecific Fab2, a bispecific camelid antibody, or a bispecific peptibody scFv-Fc, a bispecific IgG, and a knob and hole bispecific IgG, a Fc- Fab, and a knob and hole bispecific Fc-Fab.

[0123] For example, one can employ known techniques such as phage display to generate and select for small proteins having a binding domain similar to an antibody complementarity -determining region (CDR). In some embodiments, the first or second binding domain includes a scFv. In other embodiments, the first or second binding domain includes a Fab. The first binding domain can also be derived from a natural or synthetic ligand that specifically binds to an autophagosome. The second binding domain can be denved from any known or to be developed antigen binding agents, e.g.. any therapeutic antibodies, that specifically binds to target protein, whether soluble or membrane-associated.

[0124] The binding domains can include naturally-occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., binding affinity. Generally, the binding affinity of an antigen-binding moiety, e.g., an antibody, for a target antigen (e.g., PD-L1) can be calculated by the Scatchard method described by Frankel et al., Mol Immunol (1979) 16: 101-06. In some embodiments, binding affinity is measured by an antigen / antibody dissociation rate. In some embodiments, binding affinity is measured by a competition radioimmunoassay. In some embodiments, binding affinity is measured by ELISA. In some embodiments, antibody affinity is measured by flow cytometry. In some embodiments, binding affinity is measured by bio-layer interferometry.

[0125] Bispecific antibodies can be prepared by known methods. Embodiments of the disclosure include “knob-into-hole” bispecific antibodies, wherein the otherwise symmetric dimerization region of a bispecific binding agent is altered so that it is asymmetric. For example, a knob-into-hole bispecific IgG that is specific for antigens A and B can be altered so that the Fc portion of the A-binding chain has one or more protrusions (“knobs”), and the Fc portion of the B-binding chain has one or more hollows (“holes”), where the knobs and holes are arranged to interact. This reduces the homodimerization (A-A and B-B antibodies), and promotes the heterodimerization desired for a bispecific binding agent. See, e.g., Y. Xuet al., mAbs (2015) 7(l):231-42. In some embodiments, the bispecific binding agent has a knob-into-hole design.

[0126] In some embodiments, the first binding domain of the synthetic autophagy receptor provided herein comprises a natural autophagy^ receptor, e.g., p62 or a fragment thereof, and the second binding domain comprises an antibody or antigen binding fragment thereof. In some embodiments, the first binding domain of the synthetic autophagy receptor provided herein comprises an LIR motif which is fused to the second binding domain which comprises an antibody or antigen binding fragment thereof.

[0127] In some embodiments, once bound by the second binding domain, the target moiety is degraded at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% greater than a control. In one embodiment, the target protein is huntingtin, when bound by the synthetic autophagy receptor described herein, it is degraded 70% more than control.

[0128] Degradation of the target moiety can be measured using methods well know n in the art including Western Blot, HiBiT luminescence assay.Nucleic Acid Molecules

[0129] In one aspect, some embodiments disclosed herein relate to nucleic acid molecules comprising nucleotide sequences encoding the synthetic autophagy7receptors of the disclosure, including expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which direct in vivo expression of the synthetic autophagy receptors in a host cell. In some embodiments, the synthetic autophagy receptor described herein is expressed from a single genetic construct.

[0130] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally between about 5 Kb and about 50 Kb, for example between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb. between about 20 Kb and about 40 Kb. about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0131] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and enoughregulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette may be inserted into a vector for targeting to a desired host cell or tissue and / or into an individual. Thus, in some embodiments, an expression cassette of the disclosure comprises a nucleotide sequence encoding a synthetic autophagy7receptor operably linked to expression control elements sufficient to guide expression of the cassette in vivo. In some embodiments, the expression control element comprises a promoter and / or an enhancer and optionally, any or a combination of other nucleic acid sequences capable of effecting transcription and / or translation of the coding sequence.

[0132] In some embodiments, the nucleotide sequence is incorporated into an expression vector. Vectors generally comprise a recombinant polynucleotide construct designed for transfer between host cells, which may be used for the purpose of transformation, i.e., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Expression vectors further include a promoter operably linked to the recombinant polynucleotide, such that the recombinant polynucleotide is expressed in appropriate cells, under appropriate conditions. In some embodiments, the expression vector is an integrating vector, which can integrate into host nucleic acids.

[0133] In some embodiments, the expression vector is a viral vector, which further includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. Retroviral vectors contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Lentiviral vectors are viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.

[0134] The nucleic acid sequences can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.Methods for codon optimization are know n in the art. Codon usages within the coding sequence of the proteins disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0135] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule encoding the proteins disclosed herein. The expression cassette generally contains coding sequences and sufficient regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into an individual. An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, or bacteriophage, as a linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, i.e., operably linked.

[0136] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any synthetic autophagy' receptor disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York. NY: Wiley-Liss;Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase ChainReaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York. NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0137] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0138] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (S V40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Vircd Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0139] The precise components of the expression system are not critical. For example, a synthetic autophagy receptor as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult P. Jones, "Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0140] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally but encode the same gene product because the genetic code is degenerate. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be doublestranded or single-stranded (e.g., comprising either a sense or an antisense strand).

[0141] The nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of a synthetic autophagy receptor) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), transcripts can be produced, for example, by in vitro transcription.Recombinant Cells and Cell Cultures

[0142] The nucleic acid of the present disclosure can be introduced into a host cell to produce a recombinant cell containing the nucleic acid molecule. Accordingly, some embodiments of the disclosure relate to methods for making recombinant cells, including the steps of (a) providing a cell capable of protein expression and (b) contacting the provided cell with any of the recombinant nucleic acids described herein.

[0143] Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery', and the like.

[0144] Accordingly, in some embodiments, the nucleic acid molecules are delivered to cells by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA directed CRISPR / Cas9. or DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule presentin the recombinant host cell as a mini-circle expression vector for a stable or transient expression.

[0145] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0146] Lentiviral systems are also suitable for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy -target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0147] In some embodiments, host cells are genetically engineered (e.g., transduced, transformed, or transfected) with, for example, a vector comprising a nucleic acid sequence encoding a synthetic autophagy receptor as described herein, either a virus-derived expression vector or a vector for homologous recombination further comprising nucleic acid sequences homologous to a portion of the genome of the host cell. Host cells can be either untransformed cells or cells that have already been transfected with one or more nucleic acid molecules.

[0148] In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is transformed in vivo. In some embodiments, the cell is transformed ex vivo. In some embodiments, the cell is transformed in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, therecombinant cell is an immune system cell, e.g., a lymphocyte (e.g. , a T cell or NK cell), or a dendritic cell.

[0149] In another aspect, provided herein are various cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Synthesis

[0150] Synthetic autophagy receptors can be synthesized using the techniques of recombinant DNA and protein expression. For example, for the synthesis of DNA encoding a synthetic autophagy receptor of the disclosure, suitable DNA sequences encoding various autophagy receptors and antibodies are widely available. Such sequences encoding the first and second binding domains are inserted by standard methods, and the resulting nucleic acids encoding full-length synthetic autophagy receptors are transduced into suitable host cells and expressed. Alternatively, the nucleic acids can be expressed in a cell-free expression system, which can provide more control over oxidation and reduction conditions, pH, folding, glycosylation, and the like.

[0151] In some embodiments, the synthetic autophagy receptors can have two different complementary determining regions (CDRs), each specific for either the target moiety or the autophagosome. Thus, two different heavy chains and two different light chains are required. In other embodiments, the bispecific binding agents can have one or more CDRs specific for the target moiety and a binding domain (e.g.. the first binding domain) specific for the autophagosome. These may be expressed in the same host cell, and the resulting product will contain a mixture of homodimers and bispecific heterodimers. Homodimers can be separated from the bispecific antibodies by affinity purification (for example, first using beads coated with one antigen, then beads coated with the other antigen), reduced to monomers, and reassociated. Alternatively, one can employ a "‘knobs into holes” design, in which a dimerization region of a heavy chain constant region is altered so that the surface either protrudes (“knob”) from the surface (as compared to the wild type structure) or formsa cavity (“hole”) in such a way that the two modified surfaces are still capable of dimerizing. The knob heavy chain and its associated light chain are then expressed in one host cell, and the hole heavy chain and associated light chain are expressed in a different host cell, and the expressed proteins are combined. The asymmetry in the dimerization regions promotes the formation of heterodimers. To obtain dimerization, the two “monomers” (each consisting of a heavy chain and a light chain) are combined under reducing conditions at a moderately basic pH (e.g., about pH 8 to about pH 9) to promote disulfide bond formation between the appropriate heavy chain domains. See, e.g., US 8216805 and EP 1870459A1, incorporated herein by reference.

[0152] The binding activity of the synthetic autophagy receptors of the disclosure can be assayed by any suitable method know n in the art. For example, the binding activity of the synthetic autophagy receptors of the disclosure can be determined by, e.g.. Scatchard analysis (Munsen et al., Analyt Biochem (1980) 107:220-39). Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA, BIACORE® assays, Octet® BLI. and / or KENEXA® assays. A binding domain that preferentially or specifically binds (used interchangeably herein) to a target moiety or autophagosome is a term well understood in the art, and methods to determine such specific or preferential binding are also known in the art. A binding domain is said to exhibit specific or preferential binding if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity’ with a particular antigen or epitope than it does with alternative antigens or epitopes. A binding domain specifically or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or w ith greater duration than it binds to other substances. Also, a binding domain specifically or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to other substances present in the sample. It is also understood by reading this definition, for example, that a binding domain which specifically or preferentially binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, specific binding and preferential binding do not necessarily require (although it can include) exclusive binding.Pharmaceutical Compositions

[0153] In some embodiments, the synthetic autophagy receptor, nucleic acids, and recombinant cells of the disclosure can be incorporated into compositions, includingpharmaceutical compositions. Such compositions ty pically include the synthetic autophagy receptor, nucleic acids, and / or recombinant cells, and a pharmaceutically acceptable excipient, e.g., a carrier.

[0154] Synthetic autophagy7receptors of the disclosure can be administered using formulations used for administering recombinant proteins, antibodies and antibody -based therapeutics, or formulations based thereon. Nucleic acids of the disclosure are administered using formulations used for administering oligonucleotides, antisense RNA agents, and / or gene therapies such as CRTSPR / Cas9 based therapeutics.

[0155] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable earners include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that it can be administered by syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, poly alcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0156] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of theactive ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0157] In some embodiments, the synthetic autophagy receptors of the disclosure are administered by transfection or infection with nucleic acids encoding them, using methods known in the art, including but not limited to the methods described in McCaffrey et al., Nature (2002) 418:6893, Xia et al. , Nature Biotechnol (2002) 20: 1006-10. and Putnam, Am J Health Syst Pharm (1996) 53: 151-60. erratum A Am . J Health Syst Pharm (1996) 53:325.

[0158] Synthetic autophagy receptors of the disclosure can be administered using a formulation comprising a fusogenic carrier. These are carriers capable of fusing with the plasma membrane of a mammalian cell. Fusogenic carriers include, without limitation, membrane-encapsulated viral particles and carriers based thereon, exosomes and microvesicles (see, e.g., Y. Yang et al., J Extracellular Vessicles (2018) 7: 144131), fusogenic liposomes (see, e.g., Bailey et al., US 5552155; Martin et al., US 5891468; Holland et al., US 5885613; and Leamon, US 6379698).Methods of the Disclosure

[0159] The present disclosure provides, among others, a method of treating a disorder in a subject. The method includes administering to a subject in need thereof, a therapeutically effective amount of the synthetic autophagy receptor, the nucleic acid, the vector, the engineered cell, or the pharmaceutical composition provided herein. The disorder that can be treated by the various compositions described herein can be a neoplastic disorder, an inflammatory disease, metabolic disorder, an endocrine disorder, and a neurological disorder.

[0160] In some embodiments, the condition to be treated includes a neoplastic disorder. Some non-limiting neoplastic disorders that can be treated by the various compositions described herein include, without being limited to, breast cancer. B cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer.

[0161] In some embodiments, the condition to be treated includes an inflammatory disease. Some non-limiting inflammatory diseases that can be treated by the various compositionsdescribed herein include, without being limited to, inflammatory intestinal disease, rheumatoid arthritis, lupus, Crohn's disease, and ulcerative colitis.

[0162] In some embodiments, the condition to be treated includes a metabolic disorder. A metabolic disorder generally refers to a disorder that negatively alters the body's processing and distribution of macronutrients such as proteins, lipids, and carbohydrates. For example, metabolic disorders can happen when abnormal chemical reactions in the body alter the normal metabolic process. Metabolic disorders can also include inherited single gene anomalies, most of which are autosomal recessive. Further, metabolic disorders can be complications of severe diseases or conditions, including liver or respiratory' failure, cancer, chronic obstructive pulmonary' disease (COPD. includes emphysema and chronic bronchitis), and HIV / AIDS. Some non-limiting metabolic disorders that can be treated by the various compositions described herein include, without being limited to, diabetes, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Niemann-Pick, phenylketonuria (PKU), porphyria, Tay-Sachs disease, and Wilson’s disease.

[0163] In some embodiments, the condition to be treated includes an endocrine disorder. Some non-limiting neurological disorders that can be treated by the various compositions described herein include, without diabetes mellitus, acromegaly (overproduction of growth hormone). Addison's disease (decreased production of hormones by the adrenal glands), Cushing’s syndrome (high cortisol levels for extended periods of time), Graves’ disease (ty pe of hyperthyroidism resulting in excessive thyroid hormone production), Hashimoto’s thyroiditis (autoimmune disease resulting in hy pothy roidism and low production of thyroid hormone), hyperthyroidism (overactive thyroid), hypothyroidism (underactive thyroid), and prolactinoma (overproduction of prolactin by the pituitary gland).

[0164] In some embodiments, the condition to be treated includes a neurological disorder. Some non-limiting neurological disorders that can be treated by the various compositions described herein include, without being limited to, neurodegenerative disorders (e g., Parkinson's, or Alzheimer's) or autoimmune disorders (e.g., multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy / sleep disorders(including circadian rhythm disorders,insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nen e cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g. Giles de la Tourette's syndrome); Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity7resulting in neurodysthesias in diseases such as diabetes, MS and motor neurone disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject. In some exemplary embodiments, the neurological disorders encompassed herein includes Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

[0165] In some embodiments, the condition to be treated includes an infection by a pathogenic microorganism. As used herein, "‘pathogenic microorganism'’ can be a microorganism having an ability to infect a host and cause a disease. Pathogenic microorganisms can include, without limitation, bacterium, fungus, virus, yeast, cladmere, or non-normally present in a host or capable of causing a pathological condition in the host. Pathogenic microorganisms include both gram negative and gram positive bacteria. Examples of gram-negative bacteria include, without limitation. Pseudomonas aeruginosa, Vibrio cholerae, and pathogenic E. coli (0-157), and examples of gram-positive bacteria include, without limitation, Staphylococcus aureus.Administration of Synthetic Autophagy Receptors

[0166] Administration of any one or more of the therapeutic compositions described herein, e.g.. synthetic autophagy receptors, nucleic acids, recombinant cells, and pharmaceutical compositions, can be used to treat individuals having a condition described herein.

[0167] Synthetic autophagy7receptors of the disclosure are ty pically administered in solution or suspension formulation by inj ection or infusion. In another embodiment, a synthetic autophagy receptor is administered by systemic infusion.

[0168] The effective dose of the synthetic autophagy receptors can be determined by a skilled person in the field, e.g. a physician. The effective dose of any given synthetic autophagy7receptor may depend on the binding affinity7for each of the ligands, and the degree of expression of each of the ligands. The range of effective concentrations, however, can be determined by one of ordinary skill in the art, using the disclosure and the experimental protocols provided herein. Similarly, using the effective concentration one can determine the effective dose or range of dosages required for administration.

[0169] Depending on the disease or disorder to be treated, the severity and extent of the disease, the subject’s health, and the co-administration of other therapies, repeated doses may be administered. Alternatively, a continuous administration may be required.Administration of recombinant cells to an individual

[0170] In some embodiments, the methods involve administering the recombinant cells to an individual who is in need of such method. This administering step can be accomplished using any method of implantation known in the art. For example, the recombinant cells can be injected directly into the individual’s bloodstream by intravenous infusion or otherwise administered to the individual.

[0171] The terms “administering”, “introducing”, and “transplanting” are used interchangeably herein to refer to methods of delivering recombinant cells expressing the bispecific binding agents provided herein to an individual. In some embodiments, the methods comprise administering recombinant cells to an individual by a method or route of administration that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery’ to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to an individual can be as short as a few hours, e.g., twenty -four hours, to a few days, to as long as several years, or even long-term engraftment for the lifetime of the individual.

[0172] When provided prophylactically, in some embodiments, the recombinant cells described herein are administered to an individual in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant stem cell population serves to prevent the occurrence of symptoms of the disease or condition.

[0173] When provided therapeutically in some embodiments, recombinant stem cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0174] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 102cells, at least 5 x io2cells, at least 103cells, at least 5 x io3cells, at least 104cells, at least 5 x 104cells, at least 10’ cells, at least 2 x io5cells, at least 3 x io5cells, at least 4 x io5cells, at least 5 x io5cells, at least 6x IO5cells, at least 7 x io5cells, at least 8 x io5cells, at least 9 x io5cells, at least 1 x 1O6cells, at least 2 x io6cells, at least 3 x io6cells, at least 4 x io6cells, at least 5 x io6cells, at least 6 x io6cells, at least 7 x io6cells, at least 8 x io6cells, at least 9 x io6cells, or multiples thereof. The recombinant cells can be derived from one or more donors or can be obtained from an autologous source (i.e., the human subject being treated). In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.

[0175] In some embodiments, the delivery of a composition comprising recombinant cells (i.e., a composition comprising a plurality of recombinant cells a synthetic autophagy receptor provided herein) into an individual by a method or route results in at least partial localization of the cell composition at a desired site. A cell composition can be administered by any appropriate route that results in effective treatment in the individual, e.g., administration results in delivery to a desired location in the individual where at least a portion of the composition delivered, e.g., at least 1 x io4cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation, and the like. Injection modes include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion. In some embodiments, the route is intravenous. For the delivery’ of cells, administration by injection or infusion can be made.

[0176] In some embodiments, the recombinant cells are administered systemically, in other words a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, instead, the individual’s circulatory system and. thus, is subject to metabolism and other like processes.

[0177] The efficacy’ of a treatment with a composition for the treatment of a disease or condition can be determined by the skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective treatment if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some nonlimiting examples include a human, or a mammal) and includes: (1) inhibiting diseaseprogression, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g. , causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0178] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to an individual, such as one who has, is suspected of having, or is at risk for a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0179] In some embodiments, the individual is a mammal. In some embodiments, the mammal is human. In some embodiments, the individual has or is suspected of having a disease associated with cell signaling mediated by a cell surface protein (e.g., a membrane- associated target protein) or a soluble target protein. In some embodiments, the disorder is a neoplastic disorder, an inflammatory disease, and a neurological disorder.Systems and Kits

[0180] Also provided herein are systems and kits including the synthetic autophagy receptors, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein as well as written instructions for making and using the same. For example, provided herein, in some embodiments, are systems and / or kits that include one or more of: a bispecific binding agent synthetic autophagy receptor as described herein, a recombinant nucleic acid as described herein, a recombinant cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, the systems and / or kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters used to administer one any of the provided bispecific binding agents, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating an activity of a cell, inhibiting a target cancer cell, or treating a disease in an individual in need thereof.

[0181] Any of the above-described systems and kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents for in vitro production of the bispecific binding agents.

[0182] In some embodiments, a system or kit can further include instructions for using the components of the kit to practice the methods. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, and the like. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. . associated with the packaging or sub-packaging), and the like. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, and the like. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.EXAMPLES

[0183] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited above.

[0184] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1Materials and Methods

[0185] This Example describes the materials and methods used in Examples 2-9.

[0186] Plasmid construction. The plasmids in this work were constructed by Gibson assemblies of PCR-amplified DNA fragments. Deletion-mutant constructs were obtained by PCR-amplification and treatment of KLD enzy me mix. All the degraders were cloned into amammalian expression vector pcDNA3.1(+). The degraders in this study contain a C- terminal HA-epitope tag (9 a.a., YPYDVPDYA). Antibody against the ALFA-epitope tag was based on a reported amino acid sequence (Gotzke, H.; Kilisch, M.; Martinez-Carranza, M.; Sograte-Idrissi, S.; Rajavel, A.; Schlichthaerle, T.; Engels, N.; Jungmann, R.; Stenmark, P.; Opazo, F.; Frey, S. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 2019. 10, 4403) and codon optimized. A 32-a.a. linker (Gaudelli, N. M.; Komor, A. C.; Rees. H. A.; Packer, M. S.; Badran, A. H.; Bryson, D. L; Liu, D. R. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 2017, 551, 464-471) was inserted between two units of antibodies or LIR motifs. Unless stated otherwise, all the targets for degradation were cloned into a customized mammalian expression vector under the HSV promoter (pHSV vector). The pHSV vector was designed by replacing the CMV enhancer-promoter in pcDNA3.1(+) with the HSV promoter, with the start codon 35 bp downstream from the HSV promoter. All the protein targets on the pHSV vector contain an N-terminal HiBiT tag (11 a.a., VSGWRLFKKIS) and a C-terminal ALFA-tag (13 a.a., SRLEEELRRRLTE). The doxycycline-inducible constructs for representative AceTAC degraders were based on a Tet-On 3G vector (Addgene. Cat# 96930), replacing the EGFP-coding sequence with the corresponding AceTAC degradercoding sequence. The MitoAnchor construct was cloned into both the pcDNA3.1(+) vector and the pHSV vector. The rest of the Membrane-anchor constructs with a C-terminal ALFA- tag were built into the pcDNA3. 1(+) vector. The N-terminal organelle targeting sequences were originated from previous reports: mitochondria - (Addgene, Cat# 136623), peroxisome - (Addgene, Cat# 67764), ER - (Addgene, Cat# 64855), and Golgi apparatus - (Addgene.Cat# 14873). All constructs were sequence-verified by Sanger sequencing.

[0187] Cell culture. The U2OS cell line was acquired from ATCC (Cat# HTB-96). Cells were grown at 37 °C in a humidified atmosphere (5% CO2), cultured and passaged in Dulbecco's modified eagle medium + GlutaMAX (DMEM, ThermoFisher, Cat# 10567014) supplemented with 10% fetal bovine serum (FBS). Cells were passaged before reaching -70% confluency, with the media replaced every 3 days.

[0188] HiBiT luminescence assay for the quantification of targeted protein degradation. A total of 3.5 k U2OS cells in 100 pL complete DMEM medium per well was seeded in a 96-well microplate (Coming, Cat# 3610) 24-hour prior to the experiment. The complexes between 90 nL Xfect transfection reagent (Takara Bio, Cat# 631318) and 300 ng plasmids [150 ng pHSV plasmids encoding the protein target and 150 ng pcDNA3. 1(+) plasmids encoding the degrader / empty vector (EV; ThermoFisher, Cat# V79020)] weremixed in 10 pL Xfect reaction buffer for 10 min. After replacing the media with 90 pL fresh complete DMEM medium, the Xfect-plasmid mixture was added into the well and incubated for 24 hours. The medium that contained the Xfect-plasmid mixture was replaced with 200 pL fresh complete DMEM medium and incubated at 37 °C for another 6 hours. Meanwhile, the HiBiT lytic solution was prepared by diluting the HiBiT lytic substrate 1:50 and the LgBiT protein 1: 100 into a calculated volume of HiBiT lytic buffer and further mixing with the same volume of phosphate buffer (PBS, pH 7.4). Next, the DMEM mediumin each well was replaced with 150 pL HiBiT lytic solution at room temperature, followed by mixing on an orbital shaker (Benchmark Scientific, Cat# BT1502) at 600 rpm for 10 minutes. The luminescence of each well was immediately measured on a Tecan Infinite 200 PRO plate reader (Integration time: 1 second). After subtracting the value of the assay background (non-transfected U2OS cells treated with the HiBiT lytic solution), the luminescence value of the EV / target group was normalized as 100%.

[0189] Immunofluorescence (IF). A total of 8.5 k U2OS cells in 200 pL complete DMEM medium per well were cultured in an 8-well chambered cover glass (Cellvis. Cat# C8-1.5H- N) for 24 hours before the experiment. The complexes between 0.23 pL Xfect transfection reagent and 750 ng plasmids [375 ng pHSV plasmids encoding the HTT-103Q and 375 ng pcDNA3.1(+) plasmids encoding the degrader; For the control group, 750 ng pHSV plasmids for the HTT-103Q-expression] were mixed in 10 pL Xfect reaction buffer for 10 min. After replacing the media with 130 pL fresh complete DMEM medium, the Xfect- plasmid mixture was spiked into the well. After incubating for 24 hours at 37 °C, transfected cells were rinsed once with cold PBS and fixed / permeated using the BD Fixation / Permeablization Kit (BD Biosciences, Cat# 554714). After blocking with PBST S34 (PBS + 0.1% Tween-20) that contains 1% BSA and 0.3 M glycine for 30 minutes, the cells were incubated at 4 °C overnight with diluted primary antibodies. After washing the cells with PBS three times, the cells were incubated with diluted secondary antibodies for 1 hour in the dark at room temperature, followed by washing with PBS. The cells in PBS were imaged on a Nikon Ti confocal microscope with a Yokagawa CSU22 spinning disk unit. The fluorescence distribution was measured with excitation wavelengths of 405 nm (Alexa Fluor 405; HA-tagged constructs), 488 nm (Alexa Fluor 488; ALFA-tagged constructs), 561 nm (Alexa Fluor 568; p62), and 647 nm (Alexa Fluor 647; LC3B). The Pearson colocalization efficient analysis was carried out using the built-in function of the Nikon NIS- Elements AR Software.List of primary and secondary antibodies for IF (diluted in PSBT that contains 1% BSA) : Chicken anti-HA-tag antibody, Abeam. Cat# ab9111, 1 :200 diluted;Mouse anti-ALFA-tag antibody, NanoTag Biotechnologies, Cat# N1582, 1 mg mL-1 , 1:500 diluted;Guinea pig anti-p62 antibody, PROGEN, Cat# GP62-C, 1 :200 diluted;Rabbit anti-LC3B antibody, Abeam, Cat# abl92890, 1: 1000 diluted;Goat-anti-chicken secondary antibody, Alexa Fluor 405. Abeam, Cat# abl75674, 1:500 diluted; Goat-anti -mouse secondary antibody, Alexa Fluor 488, Abeam, Cat# abl 50113, 1:500 diluted; Goat-anti-guinea pig secondary antibody, Alexa Fluor 568, Abeam, Cat# abl 75714. 1:500 diluted;Goat-anti-rabbit secondary antibody, Alexa Fluor 647, Abeam, Cat# abl50083, 1:500 diluted.

[0190] Co-immunoprecipitation (co-IP). The general procedure for the immunoblotting experiments was based on our previous report (Jiang, Z. W.; Kuo, Y. H.; Zhong, M. Q.; Zhang, J. C.; Zhou, X. X.; Xing, L. J.; Wells. J. A.: Wang, Y. Z.; Arkin, M. R. Adaptor- Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions. J. Am. Chem. Soc. 2022, 744, 13218-13225). For co-IP, a total of 100 k U2OS cells in complete DMEM medium were cultured in a 6-well plate (Coming, Cat# 3516) for 24 hours prior to the experiment. On the day of transfection, the complexes between 1.5 pL Xfect transfection reagent and 5 pg plasmids [2.5 pg pcDNA3.1(+) plasmids encoding HTT-103Q and 2.5 pg pcDNA3. 1(+) plasmids encoding the EV or the degrader] were mixed in 100 pL Xfect reaction buffer for 10 min. The media were replaced with 900 pL fresh complete DMEM medium and the Xfect-plasmid mixture was added dropwise into the well to incubate at 37 °C for 24 hours. Cell lysis was conducted with the NP-40 lysis buffer and the cell debris were discarded after centrifugation. The supernatant was collected as the cell lysates.

[0191] The cell lysates (-300 pL) were mixed with 3 pL rabbit anti-ALFA-tag antibodies (for the co-IP of ALFA-tagged HTT-103Q; NanoTag Biotechnologies, Cat# N1583, 1 mg mL-1 ) (or rabbit anti-HA-tag antibodies for the co-IP of HA-tagged constructs; CST. Cat# 3724S) and incubated overnight at 4 °C. Next, part of the lysates (200 pL) were incubated with the Protein-A magnetic beads (ThermoFisher, Cat# 88845), and the leftover lysates were labeled as the INPUT group. After one-hour incubation at room temperature, the beads were collected by a magnet and eluted at 90 °C for 5 min in IX LDS samplebuffer, resulting in the elution as the co-IP portion of the corresponding group. The lysates containing the unbound proteins were collected as the UNBOUND group).

[0192] For western blotting of the INPUT and UNBOUND groups, protein levels in each group were quantified with BCA assay and diluted to approximately equal concentrations with NP-40 lysis buffer. After heating in IX LDS sample buffer at 90 °C for 5 min, equal amounts of INPUT and UNBOUND samples were loaded into lanes of a Bolt 4-12% bis-tris gel (ThermoFisher, Cat# NW04122BOX, NW04125BOX. or NW04127BOX; 90 V constant for 100 min). For co-IP samples, -15 pL elution from each group was loaded in each lane. After transferring proteins from the gel to a PVDF membrane using the iBlot-2 gel transfer system, the transferred PVDF membrane was blocked with TBS blocking buffer (LI-COR Biosciences, Cat# 927-60001) at room temperature for an hour. The membrane was probed with primary antibodies overnight at 4 °C. After washing with IX TBST (50 mM Tris HCl, 150 mM NaCl, 0.1% Tween-20) three times, the membrane was incubated in secondary antibodies for 1 h at room temperature. The membranes were washed with IX TBST imaged on an Odyssey CLx infrared imaging system (LI-COR Biosciences).List of primary and secondary antibodies for co-IP and western blots (diluted in TBS blocking buffer containing 0.1% Tween-20):Mouse anti-HA-tag antibody, CST, Cat# 2367S, 1:1000 diluted;Rabbit anti-HA-tag antibody, CST, Cat# 3724S, 1: 1000 diluted;Mouse anti-ALFA-tag antibody, NanoTag Biotechnologies, Cat# N1582, 1 : 1000 diluted; Rabbit anti-vinculin antibody, CST, Cat# 13901 S, 1 :2000 diluted;Goat-anti-rabbit secondary antibody, AzureSpectra 700 conjugates, Azure Biosystems, Cat# AC2128, 1:5000 diluted;Goat-anti-mouse secondary antibody. AzureSpectra 700 conjugates, Azure Biosystems, Cat# AC2129, 1:5000 diluted;Goat-anti-rabbit secondary antibody, AzureSpectra 800 conjugates, Azure Biosystems, Cat# AC2134, 1:5000 diluted;Goat-anti-mouse secondary antibody. AzureSpectra 800 conjugates, Azure Biosystems, Cat# AC2135, 1:5000 diluted.Autophagy modulation assays

[0193] ATG7-knockdown assay. The RNA interference of ATG7 was conducted through reverse transfection of the siRNA for ATG7 (siATG7; CST, Cat# 6604). The complexes between 0.5 pL Xfect-RNA transfection reagent (Takara Bio, Cat# 631450) and 5 pmol siATG7 [or the control siRNA (siCtrl); MilliporeSigma, Cat# SIC001] were mixed in 10 pLXfect reaction buffer for 10 min and added into a 96-well microplate (Coming, Cat# 3610). A total of 8 k U2OS cells in 90 pL complete DMEM medium per well were added into the microplate and incubated for 24 hours at 37 °C. Next, the HiBiT luminescence assay was conducted on these siRNA-transfected U2OS cells.

[0194] The knockdown of ATG7 was validated by western blots. Briefly, with the siRNAtransfection reagents scaled up from a 96-w ell microplate to a 12-well microplate, a total of 80 k U2OS cells in complete DMEM medium per well w ere added into in a 12-well microplate (Coming, Cat# 3513) for 24 hours. The siRNA-complex-containing medium was replaced with fresh complete DMEM medium and incubated for another 24 hours. Next, the western blotting of U2OS cells was conducted following the “Western blots” section.List of primary antibodies for western blots (diluted in TBS blocking buffer containing 0. 1% Tween- 20):Rabbit anti-vinculin antibody, CST, Cat# 13901S, 1 :2000 diluted;Mouse anti-ATG7 antibody, Proteintech, Cat# 67341-1-Ig, 1:500 diluted.

[0195] ATG5-knockdown assay. The RNA interference of ATG5 was conducted through reverse transfection of the siRNA for ATG5 (siATG5;lntegrated DNA Technologies, sense strand: 5’-ACGCUAAAAGGCUUACAGUAUCAGA-3’). The complexes between 0.5 pL Xfect-RNA transfection reagent (Takara Bio, Cat# 631450) and 5 pmol siATG5 [or the control siRNA (siCtrl); MilliporeSigma, Cat# SIC001] w ere mixed in 10 pL Xfect reaction buffer for 10 min and added into a 96-well microplate (Coming, Cat# 3610). A total of 5 k U2OS cells in 90 pL complete DMEM medium per well were added into the microplate and incubated for 24 hours at 37 °C. Next, the HiBiT luminescence assay was conducted on these siRNA-transfected U2OS cells.

[0196] The knockdown of ATG5 was validated by western blots. Briefly, with the siRNAtransfection reagents scaled up from a 96-well microplate to a 12-well microplate, a total of 60 k U2OS cells in complete DMEM medium per well were added into in a 12-well microplate (Coming, Cat# 3513) for 24 hours. The siRNA-complex-containing medium w as replaced with fresh complete DMEM medium and incubated for another 24 hours. Next, the western blotting of U2OS cells was conducted following the “Western blots” section.List of primary antibodies for western blots (diluted in TBS blocking buffer containing 0. 1% Tween- 20):Mouse anti-GAPDH antibody , SCBT, Cat# sc-47724, 1 :5000 diluted;Rabbit anti-ATG5 antibody, CST, Cat# 12994, 1 :1000 diluted.

[0197] Small-molecule modulation and serum-starvation assays. The HiBiT luminescence assay was conducted with modifications after plasmid transfection. For the treatment groups, complete DMEM medium containing Torinl (250 nM) or Bafilomycin-Al (100 nM), or amino acid-depleted DMEM medium (for the serum-starvation condition; USBio, Cat# D9800-13) was used to replace the transfection-complex-containing medium, followed by incubation for 6 hours at 37 °C. The WT-control group was following the original HiBiT luminescence assay procedure that uses complete DMEM medium for the 6- hour incubation. After subtracting the value of the assay background (non-transfected U2OS cells treated with the HiBiT lytic solution), the luminescence value of the EV / target group in the WT-control group was normalized as 100%. For the evaluation of the ubiquitin- proteasome pathway, the carfilzomib-treatment (200 nM) was conducted following the same protocol.

[0198] P62-knockout(p62KO) assay. Generation of p62KO-U2OS cells were generated based on our previous report for the generation of p47KO-U2OS cells (Jiang, Z. W.; Kuo, Y. H.; Zhong, M. Q.; Zhang. J. C.; Zhou, X. X.; Xing. L. J.; Wells, J. A.; Wang, Y. Z.; Arkin, M. R. Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions. J. Am. Chem. Soc. 2022, 144, 13218-13225). The p62KO-plasmid was acquired from SCBT, Cat# sc-400099. After cell-sorting and expansion, only three viable clones were obtained. Clone# C3 was verified by western blots and genomic DNA analysis using Sanger sequencing to be the p62KO U2OS clone, thus referred as the “p62-KO U2OS cell’’. The p62KO assay was then conducted with the ‘'HiBiT luminescence assay” by comparing between wildtype U2OS and p62-KO U2OS cells for the HTT - 103 Q degradation.

[0199] Western blots. The general procedure for the immunoblotting experiments was based on our previous report (Jiang, Z. W.; Kuo, Y. H.; Zhong, M. Q.; Zhang, J. C.; Zhou, X. X.; Xing, L. J.; Wells, J. A.; Wang, Y. Z.; Arkin, M. R. Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions. J. Am. Chem. Soc. 2022. 144, 13218-13225). A total of 45 k U2OS cells in 1 mL complete DMEM medium per well were cultured in a 12-well plate (Coming. Cat# 3513) for 24 hours prior to the experiment. On the day of transfection, the complexes between 0.75 pL Xfect transfection reagent and 2.5 pg plasmids [1.25 pg pHSV plasmids encoding the protein target and 1.25 pg pcDNA3. 1 (+) plasmids encoding the EV or the degrader] were mixed in 50 pL Xfect reaction buffer for 10 min. The medium was replaced with 450 pL fresh complete DMEM medium and the Xfect-plasmid mixture was addeddropwise into the well to incubate at 37 °C for 24 hours. Cell lysis was conducted with the RIPA lysis buffer (ThermoFisher, Cat# 89900) and the supernatant after centrifugation was collected as the cell lysates. The protein gel electrophoresis, protein transfer, antibody probing on PVDF membranes were following the “Co-IP” section.List of primary antibodies for western blots (diluted in TBS blocking buffer containing 0. 1% Tween-20)'.Mouse anti-LC3B antibody, CST, Cat# 83506. 1: 1000 diluted;Rabbit anti-p62 antibody, Abeam, Cat# abl09012, 1 :1000 diluted;Mouse anti-vinculin antibody, SCBT, Cat# sc-73614, 1:2000 diluted.

[0200] Quantification of targeted organelle degradation. Mitochondria. A total of 20 k U2OS cells in 500 pL complete DMEM medium per well were cultured in a 24-well microplate (Coming, Cat# 3524) for 24 hours prior to the experiment. The complexes between 0.3 pL Xfect transfection reagent and 1 pg plasmids (500 ng plasmids encoding the BFP -tagged degrader and 500 ng plasmids encoding the MitoAnchor) were mixed in 25 pL Xfect reaction buffer for 10 min. After replacing the media with 250 pL fresh complete DMEM medium, the Xfect-plasmid mixture was added dropwise into the well and incubated at 37 °C for 24 hours. Next, the transfected cells were stained with 25 nM MitoTracker-DR (ThermoFisher, Cat# M22426) in 500 pL complete FluoroBrite DMEM medium (ThermoFisher, Cat# A1896701; containing 10% FBS) at 37 °C for 15 minutes. The cells were rinsed with cold PBS and detached by TrypLE enzy me (ThermoFisher, Cat# 12604013). The fluorescence intensity within the cells was measured using flow cytometry (Beckman CytoFLEX flow cytometer) with excitation wavelengths of 405 nm (BFP) and 638 nm (MitoTracker-DR). U2OS cells without plasmid transfection and MitoTracker staining were used as the blank control group.

[0201] Peroxisome. ER. and Golgi apparatus. A total of 20 k U2OS cells in complete DMEM medium were cultured with 4 pL BacMam GFP transduction reagents (ThermoFisher, Cat# C10604, Cat# C10590, or Cat# C10592) per well in a 24-well microplate for 24 hours, resulting in the targeted organelles labeled with GFP. After cotransfecting the mPlum-taggeddegrader- and Membrane-anchor-encoding plasmids, the cells were detached for flow cytometry measurement. The fluorescence intensity within the cells was measured using flow cytometry with excitation wavelengths of 488 nm (GFP) and 638 nm (mPlum). U2OS cells without BacMam transduction and plasmid transfection were used as the blank control group.

[0202] Fluorescence imaging of organelle targeting for AceTAC degradation system. For mitochondria-related imaging, a total of 8.5 k U2OS cells in 200 pL complete DMEM medium per well were cultured in an 8-well chambered cover glass for 24 hours before the experiment. The complexes between 0.23 pL Xfect transfection reagent and 750 ng plasmids (equal amount of degrader- and Mito Anchor-encoding plasmids) were mixed in 10 pL Xfect reaction buffer for 10 min. After replacing the media with 130 pL fresh complete DMEM medium, the Xfect-plasmid mixture was spiked into the well and incubated for 24 hours at 37 °C. cells were first stained with 5 pM SNAP-Cell Oregon Green (NEB, Cat# S9102S) in complete FluoroBrite DMEM medium at 37 °C for 30 minutes. After rinsing with FluoroBrite S40 DMEM once, the cells were stained with with 25 nM MitoTracker-DR in FluoroBrite DMEM at 37 °C for 15 minutes. The cells were then rinsed once with PBS and ready for live-cell imaging in FluoroBrite DMEM. The IF experiment of LC3B was carried out at this step following the procedures in the ‘‘Immunofluorescence (IF)” section.

[0203] For the imaging of the GFP-labeled organelles, a total of 8.5 k U2OS cells in complete DMEM medium were cultured with 4.3 pL BacMam GFP transduction reagents (based on the organelle) in an 8-well chambered cover glass 24 hours prior to the experiments. After co-transfecting the degrader- and Membrane-anchor-encoding plasmids, U2OS cells were first stained with 3 pM SNAP-Cell 647-SiR (NEB, Cat# S9102S) in complete FluoroBrite DMEM medium at 37 °C for 30 minutes. After rinsing with FluoroBrite DMEM once, the cells were incubated with NucBlue Live Cell Stain (ThermoFisher, Cat# R37605) in FluoroBrite DMEM at 37 °C for 5 minutes. The cells were then rinsed once with PBS and ready for live-cell imaging in FluoroBrite DMEM (Note that cells can also be fixed at this point following the procedures in the “Immunofluorescence” section). The fluorescence imaging was carried out on a Nikon Ti confocal microscope with a Yokagawa CSU22 spinning disk unit or an ECHO Revolve Microscope. For the confocal microscope, the fluorescence distribution w as measured with excitation wavelengths of 405 nm, 488 nm, 561 nm, and 647 nm. For the Revolve microscope, the fluorescence distribution was measured in three channels: DAPI (Ex: 380 / 30 Em: 450 / 50, DM: 425), FITC (Ex: 470 / 40, Em: 525 / 50. DM:495) and CY5 (Ex: 630 / 40, Em: 700 / 75, DM: 660).

[0204] AlamarBlue cell viability assay. A total of 3.5 k U2OS cells in 100 pL complete DMEM medium per well was seeded in a 96-well microplate (Coming, Cat# 3596) 24-hour prior to the experiment. The complexes between 90 nL Xfect transfection reagent (Takara Bio, Cat# 631318) and 300 ng plasmids [150 ng pcDNA3.1(+) plasmids encoding the corresponding membrane-anchor construct and 150 ng pcDNA3.1(+) plasmids encoding thedegrader / empty vector (EV)] were mixed in 10 pL Xfect reaction buffer for 10 min. After replacing the media with 90 pL fresh complete DMEM medium, the Xfect-plasmid mixture was added into the well and incubated for 24 hours. Next, the cell vi abi 1 i ty assay was conducted with the alamarBlue HS cell viability reagent (IX diluted in the complete DMEM medium, 220 pL in total; ThermoFisher, Cat# A50101) following the protocol provided S41 by the manufacturer. After incubating at 37 °C for 80 minutes, 200-pL assaying medium from each well was transferred to a 96-well microplate (Coming, Cat# 3915) and measured on a Tecan Infinite 200 PRO plate reader (Fluorescence: Ex: 560 nm, Em: 590 nm). After subtracting the value of the assay background (alamarBlue-containing medium incubated in parallel without cells), the fluorescence value of the EV+membrane-anchor group was normalized as 100%.Evaluation of AceT AC-induced mitophagy

[0205] CCCP dose optimization. A total of 3.5 k U2OS cells in 100 pL complete DMEM medium per well was seeded in a 96-well microplate 24-hour prior to the experiment. The media were then replaced with fresh complete DMEM growth medium that contained calculated concentrations of CCCP (CCCP, Cat# sc-202984). The CCCP concentrations were tested from 200 pM with 2-fold serial dilutions. Cells were incubated with the CCCP- containing medium for 24 hours at 37 °C. Next, the cellular metabolic activity was assessed by the AlamarBlue assay following the procedures described in Section 2.10. AlamarBlue cell viability assay, with a change in the incubation time for the AlamarBlue reagent from 80-min to 150-min.

[0206] Next, the mitochondrial morphology of U2OS cells treated with selected CCCP concentrations was evaluated by live cell imaging. Briefly, a total of 8.5 k U2OS cells in 200 pL complete DMEM medium per well were cultured in an 8-well chambered cover glass 24 hours prior to the experiments. After treatment with selected CCCP concentrations in complete DMEM growth medium at 37 °C for 24 hours, the cells were rinsed once with PBS, followed by staining with the MitoTracker Deep Red (25 nM) and the NucBlue Live Cell Stain (2 drops per 1 mL complete FluoroBrite DMEM medium) simultaneously at 37 °C for 15-min. The cells were then rinsed once with PBS and ready for live-cell imaging in FluoroBrite DMEM. The fluorescence imaging was carried out on a Nikon Ti confocal microscope with a Yokagawa CSU22 spinning disk unit. For the confocal microscope, the fluorescence distribution was measured with excitation w avelengths of 405 nm and 647 nm.

[0207] Western blots of autophagy / mitophagy-related proteins. A total of 100 k U2OS cells in 2 mL complete DMEM medium per well w ere cultured in a 6-well plate (Coming,Cat# 3516) for 24 hours prior to the experiment. For the CCCP-treatment group, the medium was replaced with 1 mL fresh complete DMEM growth medium that contains 25 pM CCCP. For the transfection-groups, on the day of transfection, the complexes between 1.5 pL Xfect transfection reagent and 5 pg plasmids (EV-only group; for the co-transfection groups, 2.5 pg of each plasmid was included) w ere mixed in 100 pL Xfect reaction buffer for 10 min. The media were replaced with 1 mL fresh complete DMEM medium and the Xfect-plasmid mixture was added dropwise into the well to incubate at 37 °C for 24 hours. Cell lysis was conducted with the RIPA lysis buffer (ThermoFisher, Cat# 89900) and the supernatant after centrifugation was collected as the cell lysates. The protein gel electrophoresis, protein transfer, antibody probing on PVDF membranes were following the “Co-IP"’ section.List of additional primary antibodies for western blots (diluted in TBS blocking buffer containing 0.1% Tween-20):Mouse anti-BNIP3 antibody, Proteintech, Cat# 68091-1-Ig, 1 :500 diluted; Mouse anti-NIX antibody, Proteintech, Cat# 68118-1-Ig, 1:500 diluted;Rabbit anti-TAXIBPI antibody, Proteintech. Cat# 14424-1-AP, 1 : 1000 diluted;Rabbit anti-BCL2L13 antibody, Proteintech, Cat# 16612-1-AP, 1 : 1000 diluted;Rabbit anti-FKBP8 antibody, Proteintech, Cat# 11173-1-AP, 1 : 1000 diluted;Rabbit anti-OPTN antibody, Proteintech, Cat# 10837-1-AP, 1: 1000 diluted;Rabbit anti-NDP52 antibody, Proteintech, Cat# 12229-1-AP, 1: 1000 diluted;Rabbit anti-NBRl antibody, Proteintech, Cat# 16004-1-AP, 1 : 1000 diluted; Rabbit anti-PINKl antibody, Proteintech, Cat# 23274-1-AP, 1 : 1000 diluted; Mouse anti-TOMM40 antibody, Proteintech, Cat# 66658-1-Ig, 1 : 1000 diluted; Mouse anti-MFN2 antibody, Proteintech, Cat# 67487-1-Ig, 1: 1000 diluted;Rabbit anti-ULKl antibody, CST, Cat# 8054, 1: 1000 diluted.

[0208] RT-qPCR. A total of 40 k U2OS cells in 1 mL complete DMEM medium per well were cultured in a 12-well plate for 24 hours prior to the experiment. On the day of transfection, the complexes between 0.75 pL Xfect transfection reagent and 2.5 pg plasmids [1.25 pg pHSV plasmids encoding the protein target and 1.25 pg pcDNA3. 1(+) plasmids encoding the EV or the degrader] were mixed in 50 pL Xfect reaction buffer for 10 min. The media were replaced with 500 pL fresh complete DMEM medium and the Xfect-plasmid mixture was added dropwise into the well to incubate at 37 °C for 24 hours. Next, the cells were washed once with cold PBS and detached by try psin. The RNA extraction was conducted using the Quick-RNA Miniprep Plus Kit (Zymo Research, Cat# R1057) following the manufacturer’s protocol. The extracted RNA was quantified by a microvolumespectrophotometer (Thermo Scientific NanoDrop One / Onec). Next, reverse transcription was carried out (Zymo Research, Cat# R3012) using a same amount of RNA from each group to obtain cDNA as the template for qPCR. For qPCR, the RT-product was diluted 10 times as the cDNA template. TaqMan probes and primers (details below) were ordered from Integrated DNA Technologies. The real-time PCR was performed on a QuantStudio 5 Real- Time PCR System (Applied Biosystems) with Luna Universal Probe qPCR mastermix (NEB. Cat# M3004X) using the manufacturer’s recommended protocol. Four technical replicates were carried out and the resulted average Cq value was used as one biological replicate. For each experiment, three biological replicates of RT-qPCR analysis were performed.List of primers and TaqMan probes:Forward primer for ALFA-tagged HTT-103Q: 5’-TGGCGGCTGTTCAAGAAGAT-3’; Reverse primer for ALFA-tagged HTT-103Q: 5’-CTTTTGAGGGACTCGAAGGC-3’; Probe for ALFA-tagged HTT-103Q: 5’-TCCGGTATGGCGACCCTGGAAA-3’ (5'FAM / ZEN / 3'IBFQ format);Forward primer for ALFA-tagged a-synuclein: 5’-ACAACGAGGCCTACGAGAT-3’; Reverse primer for ALFA-tagged a-synuclein: 5’-TCAGTCTTCTTCTCAGCTCCTC-3’; Probe for ALFA-tagged a-synuclein: 5 -CGAAGAGGGCTACCAGGACTACGA-3’ (5'FAM / ZEN / 3'IBFQ format);Forward primer for HPRT1 reference gene: 5’-GCGATGTCAATAGGACTCCAG-3’; Reverse primer for HPRT1 reference gene: 5 -TTGTTGTAGGATATGCCCTTGA-3’; Probe for HPRT1 reference gene: 5’-AGCCTAAGATGAGAGTTCAAGTTGAGTTTGG- 3’ (5'Cy5 / TAO / 3'IBRQ format).

[0209] Doxycycline-inducible expression for AceTAC degraders. The U2OS cells for doxycycline (Dox)-related experiments were cultured and passaged in DMEM (ThermoFisher, Cat# 10567014) supplemented with 10% tetracycline-negative FBS (GeminiBio, Cat# 100-800). The HiBiT luminescence assay was conducted following the Section ‘‘HiBiT luminescence assay for the quantification of targeted protein degradation’’. Instead of the pcDNA3. 1(+) vector, the Tet-On 3G vectors for Dox-inducible expression of AceTAC degraders were cotransfected with the HTT-103Q-encoding plasmid. Varied concentrations of Dox (Sigma- Aldrich, Cat# D9891) w ere presented at the start of the transfection process. After the plasmid-transfection into U2OS cells for 24 hours, the plasmid-Xfect complex-containing media were replaced with fresh DMEM growth medium that contained the corresponding concentration of Dox. The cells were further incubated 6hours at 37 °C, followed by the HiBiT luminescence assay readout. After subtracting the value of the assay background (non-transfected U2OS cells without Dox-treatment), the luminescence value of the EV / HTT-103Q group without Dox-treatment was normalized as 100%.EXAMPLE 2Engineered Antibody-fusion Degraders Lead to Efficient Degradation of Huntingtin Protein with Polyglutamine (polvO) Expansion

[0210] The first generation of our Ab-fusion degraders were designed by directly linking target-specific antibodies to the C-terminus of full-length p62 (p62FL). We hypothesized that keeping the wild-type structure of p62 would minimally disturb the function of p62 during selective autophagy, while accumulating the target of interest into the autophagic process through the interaction between the antibody and the target. We employed huntingtin protein with polyQ expansion as the degradation target, with an N-terminal HiBiT peptide tag and a C-terminal ALFA-epitope tag (HTT-103Q). The N-terminal HiBiT tag allowed the quantification of HTT-103Q level upon the addition of LgBiT protein and luminescence substrate (FIG. 1A).18The C-terminal ALFA-tag was presented as the recognition site for the high affinity’ nanobodies (KD ~ 26 pM19) within the AceTAC degraders. Both the degrader-encoding plasmid and the target-encoding plasmid w ere cotransfected into human osteosarcoma (U2OS) cells to assess the efficiency of targeted degradation.

[0211] In the degrader design, we also incorporated antibody tandem repeats to evaluate the effect of multivalency between the degrader and the target (FIG. 1A). In the HiBiT luminescence assay, the cells in control (EV) group were co-transfected with the protein target-encoding plasmid and an empty vector. The luminescence value from the EV group was normalized to 100%. All three p62FL-based Ab-fusion degraders (p62FL-An, where n represents the number of antibody units) demonstrated more than 50% degradation of HTT- 103Q when compared with the EV-control group (FIG. IB). Among these three degraders, p62FL-A2 induced the highest degradation efficiency (-65%) for HTT-103Q. The overexpression of p62 (p62-O / E) resulted in less than 15% degradation of HTT-103Q. indicating that the specific binding to HTT-103Q via the antibody in the AceTAC degraders w as required for efficient degradation. The successful targeted degradation of HTT-103Q bythese p62FL-Ab-fusion degraders indicated feasibility of AceTAC for targeted protein degradation, albeit with incomplete efficiency.EXAMPLE 3The LIR Motif of TP53INP2 in the AceTAC Degrader Improves the Targeted Degradation Efficacy

[0212] We sought to determine the critical domains and motifs of the AceTAC degrader that affected its targeted degradation capability. Protein engineering of the AceTAC degrader evaluated (a) deletion mutants of p62 and (b) replacement of the LIR motif. For the deletion mutants, truncations of five domains / motifs were separately generated based on p62FL-A3. Previous reports have shown the participation of these five p62 domains / motifs during selective autophagy (FIG. 1C). Apart from the described function of the LIR motif and UBA domain, (1) The Phox and Beml (PB1) domain is responsible for mediating the selfoligomerization of p62 and facilitating the co-aggregation with autophagosomal substrates;20(2) the ZZ-type zinc finger (ZZ) domain recognizes specific autophagosomal substrates via the N-degron pathway;21(3) the nuclear export signal (NES) motif modulates the nucleocytoplasmic shuttling of p62.22Surprisingly, four of the truncations (p62FL-A3AZZ. p62FL-A3ALIR, p62FL-A3ANES, and p62FL-A3AUBA) increased the degradation of HTT-103Q by at least 13% (FIG. ID). Deletion of the PB1 domain had no effect, though it has been shown to be important for endogenous autophagy.23Such a result indicated that the PB1 domain for AceTAC may not have been as important as it was for wildtype p62 during the autophagy process.

[0213] We next evaluated the effect of LIR-motif variations in the p62FL-A3 degrader on targeted protein degradation. Previous studies have tested the binding between LC3 / GABARAP proteins and the LIR motifs from various binding partners.24Based on the report, we picked three LIR motifs (TP53INP2, TBC1D25, and FYCO1) that tightly and broadly bound with six LC3 / GABARAP proteins. After replacing the p62-LIR motif in the p62FL-A3 degrader with the TP53INP2-LIR motif, the targeted degradation efficiency for HTT-103Q was significantly improved by -26%, resulting in -72% degradation of HTT- 103Q when compared to the EV group (FIG. IE). The improved degradation efficiency might have been due to the LIR motif of TP53INP2 binding the most efficiently with all six LC3 / GABARAP proteins when com-pared to 33 other tested LIR motifs.24Meanwhile, the engineered degrader that contained either TBC1D25-LIR or FYCO1-LIR achieved -10%more degradation of HTT-103Q than the one with p62-LIR, i.e., p62FL-A3. Although the absence of the p62-LIR motif (i.e., the p62FL-A3ALIR group) still surprisingly resulted in the degradation of HTT-103Q (FIG. ID), the addition of efficient LIR motifs in the degrader could significantly increase its degradation efficiency. These comparisons demonstrated that the LIR motif on the degrader could modulate its targeted degradation efficacy.EXAMPLE 4Multivalent Constructs of the AceTACs Further Enhance the Targeted Degradation Efficacy

[0214] Based on degradation results by the deletion mutants and the LIR variations, we constructed two more generations of the Ac-eTAC degraders (FIG. IF). The design principle of these new degraders centered on the repeats of the TP53INP2-LIR motif and the antibody. In detail, the second generation of AceTACs were built on p62AUBA with its LIR motif replaced by TP53INP2-LIR motif(s), followed by antibody unit(s) on its C-terminus (TmAn, where m represents the number of TP53INP2-LIR motifs, n represents the number of antibody repeats). The third generation of AceTACs was a simplified version of the second generation - it only contained repeats of the LIR motif and the antibody (AN-TmAn) (FIG. 2)

[0215] To evaluate the differences between these two generations of AceTACs. we stepwise generated the deletion mutants of T3A3, including T3A3APB1 , T3A3APB1 AZZ, and ANTS A3. Consistent with the deletion mutants of p62FL-A3 (FIG. ID), the deletion of the PB1 domain in T3A3 did not significantly affect the degradation performance (FIG. 3). Improved HTT-103Q degradation efficacy was achieved by AN-T3A3 when compared to T3A3. For the overall performance of these two generations of AceTAC degraders, at least 80% of HTT-103Q degradation was achieved (FIG. 1G-1H). However, no obvious trend of targeted degradation was ob-served to correlate wi th the number of LIR motifs or antibody units. Among these degraders, AN-T3A3 consistently achieved -90% targeted degradation of HTT-103Q. The high efficiency of AN-TmAn degraders for targeted HTT-103Q degradation suggested that the p62 domains and motifs may not be necessary for the AceTAC degradation system. Only the LIR motif and the target-specific antibody were retained in the third generation AceTAC degraders. Additionally, the ALFA-tagged HTT- 103Q mRNA level remained similar in the presence of three generations of AceTAC degraders (FIG. 4A). The targeted degradation of HTT-103Q could also be modulated in adoxycycline-inducible expression system for these three generations of AceTAC degraders (FIG. 5) These results confirmed that the degradation was induced at the protein level.EXAMPLE 5AceTAC is Applicable for the Targeted Degradation of Various Aggregation-prone Proteins

[0216] After engineering the AceTAC degraders with HTT-103Q as the model target, we tested selected AceTAC degraders against representative aggregation-prone proteins. These disease-relevant targets included wild-type a-synuclein and tau, as well as mutants of TDP- 43, fused-in-sarcoma (FUS), and tau. We replaced HTT-103Q with the protein of interest in the target-encoding plasmid, keeping the N-terminal HiBiT tag and the C-terminal ALFA- tag. For these five different protein targets in U2OS cells, AN-T1 A2 induced at least 80% degradation when compared to the EV group (FIG. 6A-6E). Consistent with HTT-103Q, the third generation of AceTAC degraders resulted in the highest targeted degradation performance (FIGs. 7A-7E). Meanwhile, in the absence of AceTAC degraders, overexpression of p62 again led to modest degradation (< 35%) of the targeted protein through enhancement of autophagy. We also confirmed the degradation results of these protein targets through western blots, which agreed with the HiBiT luminescence assays (FIG. 6F, FIGs. 8A-8E). The successful targeted degradation of several aggregation-prone proteins demonstrated the broad applicability of the AceTAC degraders.EXAMPLE 6Selective Degradation of Mitochondria is Tunable Through the AceTAC Degradation System

[0217] After the successful targeted degradation of proteins, we sought to explore whether the AceTAC system could be extended to organelles. The targeted degradation of organelles is challenging due to the large size and interfacial area of the target. Our essential design principle was to provide multiple recognition sites on the outer membrane of the organelle of interest, followed by the presence of AceTAC degraders to initiate the degradation process (FIG. 9A). We designed a SNAP -tag protein-based construct to decorate the outer mitochondrial membrane (termed MitoAnchor). The MitoAnchor contained an N-terminal Tom20 peptide for mitochondrial localization25and a C-terminal ALFA-tag to bind with the AceTAC degraders. Meanwhile, to enable the visualization of Ac-eTAC degraders in live cells, we tagged a blue fluorescent protein (BFP) to the N-terminus of the AN-TmAndegraders, denoted as the BFP-TmAn series (FIG. 10). After co-transfecting the BFP-T3A3- encoding plasmid and the MitoAnchor-encoding plasmid for 24 hours, we stained the live U2OS cells with MitoTracker Deep Red FM (MitoTracker-DR) to monitor the overall cellular mitochondrial level. Different cell populations were presented due to the nature of the transient expression. When both the BFP-T3A3 degrader and the MitoAnchor were present, the cells displayed an aggregated morphology of the mitochondrial network (FIG. 9B, cell# 1 ). In contrast, in cells where BFP-T3A3 was not expressed, mitochondria showed the regular tubular network (FIG. 9B, cells#2 and #3). Similar results were observed in the co-presence of BFP-T1A1 and MitoAnchor (FIG. 11). Such aggregation behavior resembled the process of mitochondrial autophagy (i.e., mitophagy),26consistent with the initiation of selective mitochondrial degradation through our AceTAC system.

[0218] Next, we quantified the reduction of mitochondrial contents by AceTAC -induced mitochondrial degradation through flow cytometry. To tune the level of MitoAnchor on the outer mitochondrial membrane, we engineered the MitoAnchor construct downstream of either the strong cytomegalovirus (CMV) promoter or the weak herpes simplex virus- thymidine kinase (HSV) promoter (FIG. 12).27After co-transfecting BFP-T3A3 and the MitoAnchor for 24 hours in U2OS cells, we compared the intensity of MitoTracker-DR between the BFP-T3A3(+) and the BFP-T3A3(-) populations (FIG. 9C-9D). When the MitoAnchor was under the CMV promoter for high-level expression, MitoTracker-DR intensity was reduced -33% in the BFP-T3A3(+) population when compared to the BFP- T3A3(-) population. By contrast, MitoTracker-DR intensity was reduced only -13% when the MitoAnchor was expressed under the weaker HSV promoter. Since the expression of BFP-T3A3 was kept constant under the CMV promoter, we conclude that the higher number of recognition sites on the targeted organelles led to the increased degradation by the AceTAC degraders. Apart from the tunability offered by the MitoAnchor expression, we also tested the BFP-TmAn degraders for the targeted degradation of mitochondria. Under the high-level expression of MitoAnchor (CMV promoter) in U2OS cells, all six BFP-TmAn degraders induced at least 30% reduction of the cellular MitoTracker-DR intensity, with both BFP-T1A2 and BFP-T2A2 showing -40% reduction (FIG. 9E). Viability assays using alamar blue indicated that the mitochondrial loss was not caused by cytotoxicity (FIG.13A). Moreover, when the TP53INP2-LIR motif was removed from the BFP-T1A1 degrader (i.e., BFP-A1 construct), the co-presence of BFP-A1 and MitoAnchor led to negligible degradation of mitochondria (FIG. 14A). These BFP-based control constructs (BFP-ACtrl, BFP-TlACtrl, and BFP-A1) were either unable to co-localize with the MitoAnchor or failedto induce the aggregation of mitochondria (FIG. 15). Together, these results confirmed the feasibility and tunabili ty of targeted mitochondrial degradation through the AceTAC degradation systems.EXAMPLE 7The AceTAC Degradation System is Applicable to the Degradation of Different Organelles

[0219] We envisioned that the AceTAC degradation system could be expanded to target diverse organelles in mammalian cells. Therefore, we designed a series of Membrane-anchor constructs by replacing the mitochondrial localization sequence in the MitoAnchor with a targeting sequence for the organelle of interest (FIG. 16A). Unlike mitochondria, most other organelles do not have a well-developed library of small-molecule dyes for specific staining and quantification. Therefore, we utilized a baculovirus-transduction platform (the BacMam system28) to generate cells with green fluorescent protein (GFP) labelled on the organelle of interest, taking advantage of the GFP fluorescence to quantify the level of the targeted organelle. Because GFP was not the direct target of the AceTAC degraders, the level of GFP reasonably represented the level of the labeled organelle. To avoid spectral overlap, we also replaced BFP with mPlum fluorescent protein, generating a new series of fluorescent AceTAC degraders that were denoted as the mPlum-TmAn series.

[0220] We selected peroxisome, ER, and the Golgi apparatus as the targeted organelles for degradation. For the Membrane-anchor constructs, we employed the N-terminal residues of peroxisomal biogenesis factor-3 (PEX),29cytochrome P450 (CYP45O),30and nitric oxide synthase 3 (NOS3)?1as the localization tag for peroxisome, ER, and Golgi, respectively. To evaluate the targeted degradation of each organelle, we first obtained the U2OS cells with GFP labeled on the targeted organelles through the BacMam system. Next, after cotransfecting the corresponding Membrane-anchor construct and the mPlum-TmAn-degrader construct, we compared the organelle-GFP intensity7betw een the mPlum-TmAn(+) and the mPlum-TmAn(-) cell populations through flow cytometry. For the targeted degradation of peroxisome. -35% peroxisomal GFP signal reduction was achieved using the mPlum-TlAl degrader (FIG. 16B FIG. 13B, FIG. 14B, FIG. 17A-17B). For ER degradation, -25% ER- GFP reduction w as achieved by three mPlum-based degraders (FIG. 16C, FIG. 13C, FIG. 14C, FIG. 17C-17D). The rest of the degraders show ed some ability to reduce both peroxisomal-GFP and ER-GFP. However, the Golgi-GFP intensity7change was negligible in the presence of all AceTAC degraders (FIG. 17E-17F, FIG. 18).

[0221] We assessed the cellular localization of the AceTAC degradation system for targeted organelle degradation by fluorescence microscopy. For all three tested organelles, the localization of the mPlum-Tl Al degrader varied by the distribution of the specific Membrane-anchor that was co-transfected (FIG. 16D). This again indicated that the mPlum- T1 Al degrader bound with the C-terminal ALFA tag on the Membrane-anchor construct, while the localization of the Membrane-anchor depended on its N-terminal targeting sequence. The colocalization between the Membrane-anchor / mPlum-TlAl pair and the organelle-GFP was observed for peroxisome, ER, and Golgi. However, unlike the AceT AC- induced mitochondrial degradation, the colocalization did not appear as distinct cellular aggregates for these three organelles, presumably due to the unique nature of each organelle (e.g., organelle size, homogeneity) during degradation.1, 32-34Particularly regarding the Golgi apparatus, although negligible degradation was observed from flow cytometry analysis, the live cell imaging demonstrated a clear reduction in the intensity of the Golgi juxtanuclear structure in the presence of the Golgi Membrane-anchor and the mPlum-Tl Al degrader, indicating that Golgi morphology was disrupted (FIG. 16D). Similar to the AceT AC- induced mitochondrial degradation (FIG. 15), the mPlum-based control constructs (mPlum- ACtrl, mPlum-Tl ACtrl, and mPlum-Al) did not efficiently alter the morphology of these organelles (FIG. 19, FIG. 20, FIG. 21). Overall, the AceTAC system revealed a tunable and promising strategy for targeted organelle degradation.EXAMPLE 8The Targeted Degradation by AceTAC Degraders is Facilitated by Autophagy

[0222] We assessed whether the targeted degradation by AceTAC degraders utilized the autophagy process. First, we conducted immunofluorescence (IF) to verify the cellular localization of the degrader and protein target, along with endogenous p62 and LC3B, since both p62 and LC3B have been used as common biomarkers for autophagosomes. Two representative degraders, T3A3 and AN-T3A3, were compared with the control group that was only transfected with HTT-103Q in U2OS cells. In the absence of the degrader, the HTT-103Q spread over the cytoplasm as amorphous aggregates, while it minimally colocalized with p62 and LC3B. Comparatively, in the presence of either T3A3 or AN-T3A3 degrader, the morphology of HTT-103 changed to puncta within the cytoplasm (FIG. 22A, FIG. 23, FIG. 24). Most importantly, the introduction of the AceTAC degraders led to the colocalization among the degrader itself, the degradation target (HTT-103Q), endogenous p62, and endogenous LC3B within puncta (FIG. 22B). Constructs based on controlnanobodies (ACtrl and AN-TlACtrl) failed to induce the puncta formation of HTT-103Q (FIG. 25). Interestingly, the removal of the LIR-motif in representative AceTAC degraders still led to similar puncta formation and colocalization signature as the complete AceTAC degraders (FIG. 26). Considering the efficient degradation capability of these ALIR constructs (FIG. ID), the induced aggregation step was further validated to be essential in the AceTAC-induced protein degradation. Moreover, HTT-103Q co-immunoprecipitated with the AceTAC degrader (i.e., T3A3 or AN-T3A3) from U2OS cell lysates (FIG. 22C, FIG. 27), demonstrating the binding between the target and the AceTAC degraders.

[0223] We also validated the mechanism of action for the AceTAC-induced mitophagy through IF. The colocalization among the BFP-T3A3 degrader, the MitoAnchor, LC3B, and the MitoTracker-DR occurred as an aggregated cytoplasmic structure (FIG. 22E). consistent with colocalization observed during the targeted degradation of HTT-103Q. Next, we compared the AceTAC-induced mitophagy with CCCP, a small-molecule inducer of mitophagy35(FIG. 28). We conducted western blotting of 16 proteins that were relevant to autophagy / mitophagy, including selected autophagy receptors. ATG proteins, and kinases (FIG. 29). The AceTAC degradation system decreased the level of OPTN, an autophagy receptor that was found to be involved during the mitophagy of damaged mitochondria.36Although both the AceTAC- and CCCP-induced mitophagy increased the LC3-II / I ratio, a different protein expression pattern was observed. For example, p62 was significantly increased in the AceTAC degradation system while it was decreased by the CCCP- treatment; the PINK1 level was boosted by CCCP-treatment as previously reported37whereas minimal change resulted from the AceTAC system. The reason for these differences will be further investigated.

[0224] We further investigated the mechanism of action for the AceTAC degradation system through several orthogonal validations. ATG7 is required for the formation of LC3- II,38the lipidated LC3 that anchors to the autophagosomal membrane. ATG7 -knockdown (ATG7-KD) therefore inhibits autophagic flux. We achieved more than 90% knockdown of ATG7 using siRNA (FIG. 30). When compared to wild-type U2OS cells, the degradation of HTT-103Q by the AceTAC degraders was suppressed in the cells with reduced ATG7-level (FIG. 22D) Moreover, ATG5-KD39 also decreased the degradation efficiency of AceTAC degraders against HTT-103Q (FIG. 31). The targeted degradation efficiency of AceTAC degraders also slightly increased under autophagy -inducing conditions (Torinl -treatment40and serum-starvation41) (FIG. 32). The presence of TP53INP2-LIR motif in the degrader not only enabled the binding between the degrader and the autophagosomal membrane (FIG.22A-22C), but also increased autophagic activity even in the absence of target-specific antibody based on immunoblotting for LC3II / I ratio and p62 levels (FIG. 33). This result is reasonable as the cytoplasmic presence of truncated TP53INP2 has been show n to trigger the LC3-II production and autophagosome formation.42Furthermore, we assessed if the ubiquitin-proteasome system (UPS) contributed to the AceTAC degraders by treatment of carfilzomib, an irreversible proteasome inhibitor.43Upon carfilzomib-treatment, no significant difference was observed between the degraders and the control group for HTT- 103Q degradation (FIG. 34). However, the slight differences among the three generations of AceTAC degraders suggested that we should continue to evaluate a role for the UPS in later generations of the AceTAC degradation system.

[0225] Last, to our surprise, the targeted degradation efficiency of HTT-103Q by AceTAC degraders was improved in p62-knockout (p62KO) U2OS cells compared to wildtype U2OS cells (FIG. 35). Previous studies have demonstrated that the depletion of p62 led to increased cy toplasmic HTT-polyQ proteins.44As the autophagy machinery' functions in the cytoplasm, the increased degradation efficiency’ of HTT-103Q by AceTAC degraders in p62KO cells might be attributed to the enhanced availability of protein targets (i.e., HTT- 103Q) in the cytoplasm. All these results together strongly supported that the targeted degradation by AceTAC degraders was facilitated by autophagy'.Discussion of Examples

[0226] We have described the development of Ab-fusion (AceTAC) degraders for targeted degradation of protein aggregates and organelles. These AceTAC degraders w ere inspired by the structure and function of p62, a maj or autophagy receptor. Through protein engineering, the optimized AceTAC degraders were simple constructs containing the LC3- interacting region of TP53INP2 and an ALFA-targeting nanobody, resulting in efficient degradation of intracellular proteins. When recognition sites were decorated on the outer membrane of a specific organelle, the AceTAC degradation system could be systematically tuned to induce aggregation and degradation of the targeted organelle.

[0227] Thus, the AceTAC degradation technology may be suitable for removing large-sized intracellular contents that are processed for degradation via autophagy. While several recent reports utilized the autophagy7process for targeted protein degradation, it is still challenging to develop a systematic strategy for efficient targeted organelle degradation. For example, the AUTOTAC technology utilized bifunctional small-molecules to link between the p62- ZZ domain and the target, efficiently degrading protein targets, although not exploring theorganelle context,15while Li and coworkers showed the degradation of fluorescently labeled peroxisomes in plants.45Here, we demonstrate initial inroads into targeted organelle degradation in mammalian cells, particularly for endogenous mitochondria, revealing applicable design principles for successful degradation of peroxisomes and ER. This AceTAC degradation system is built on the interaction between an epitope tag and its corresponding single-domain antibody fragment. This use of antigen / antibody recognition is highly general, but the method can also be extended to other protein-protein interaction pairs. In addition to existing antibodies, discovery methods such as phage display46will further extend the applicability of the technology, taking advantage of disease-relevant biomarkers to improve the efficiency and specificity to endogenous protein targets. Overall, the precision of the antibody-based AceTAC system possesses potential as a tool to study protein aggregates and organelle degradation, as well as drug discovery platforms. The applicability' of the AceTAC degradation system can be further broadened by the maturation of protein deliver}' methods.

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Claims

CLAIMSWHAT IS CLAIMED IS:

1. A synthetic autophagy receptor comprising: a) a first binding domain that specifically binds to an autophagosome, and b) a second binding domain that specifically binds to a target moiety, wherein the binding of the first binding domain to the autophagosome can result in the degradation of the target moiety bound to the second binding domain.

2. The synthetic autophagy receptor of claim 1, wherein the first binding domain is selected from the group consisting of natural ligands or a fragment, derivative, or small molecule mimetic thereof, antibodies and antigen binding fragments thereof.

3. The synthetic autophagy receptor of any preceding claim, wherein the first binding domain specifically binds to a protein selected from the group LC3 A, LC3B, LC3C, GAB ARAP, GABARA0L1, and GAB ARAP L2 on the autophagosome.

4. The synthetic autophagy receptor of any preceding claim, wherein the first binding domain comprises an LC3-interacting region (LIR).

5. The synthetic autophagy’ receptor of any preceding claim, wherein the LIR is derived from a protein selected from the group ATG13. FYCOL NBR1, Nix / Bnip3L, p62. TP53INP1, TP53INP2 / DOR, ULK1, ULK2, Optineunn, FUNDCI, TBC 1D5 LIR1, TBC1D5 LIR2, c-Cbl, 0-catenin, Dvl2, MAP15K, Bnip3, ATG4B, Taxlbpl / T6BP, Stbdl, NDP52, Calreticulin, Clathrin HC, FIP200 and TBC1D25.

6. The synthetic autophagy' receptor of claim 5, wherein the LIR is selected from p62, TP53INP2, FYCO1, and TBC1D25.

7. The synthetic autophagy’ receptor of claim 5, wherein the LIR is from p62.

8. The synthetic autophagy' receptor of claim 5, wherein the LIR is from TP53INP29. The synthetic autophagy’ receptor of any preceding claim, wherein the second binding domain comprises one or more antibodies.

10. The synthetic autophagy' receptor of any preceding claim, wherein the target moiety comprises a protein, protein aggregate, protein fragment, organelle, or membraneless organelle.

11. The synthetic autophagy' receptor of claim 10, wherein the target moiety comprises a protein aggregate.

12. The synthetic autophagy' receptor of claim 11, wherein the protein aggregate comprises Ap, FUS. a-synuclein, tau, TDP-43, and / or huntingtin.

13. The sy thetic autophagy' receptor of claim 10, wherein the target moiety comprises an organelle.

14. The synthetic autophagy’ receptor of claim 13, wherein the organelle is selected from the group consisting of mitochondria, peroxisome, Golgi, and endoplasmic reticulum.

15. The synthetic autophagy’ receptor of claim 14, wherein the organelle is decorated with SNAP -tag based protein constructs.

16. A nucleic acid that encodes the synthetic autophagy receptor of any one of the preceding claims.

17. The nucleic acid of claim 16, wherein the nucleic acid is operably connected to a promoter.

18. An engineered cell capable of protein expression comprising the nucleic acid of claim 16 or 17.

19. A method for making a synthetic autophagy’ receptor, the method comprising: i) providing a cell capable of protein synthesis, comprising the nucleic acid of claim 16 or 17; and ii) inducing expression of the synthetic autophagy receptor.

20. A vector, comprising the nucleic acid of claim 16 or 17.

21. The vector of claim 20, further comprising a promoter, wherein the promoter is operably linked to the nucleic acid.

22. A pharmaceutical composition, comprising the synthetic autophagy receptor, the nucleic acid, the vector, the engineered cell, of any one of the preceding claims, and a pharmaceutically acceptable excipient.

23. A method of treating a disorder in a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the synthetic autophagy receptor, the nucleic acid, the vector, the engineered cell, or the pharmaceutical composition of any one of the preceding claims.

24. The method of claim 23, wherein the disorder comprises a neurological disorder.

25. The method of claim 24, wherein the neurological disorder comprises Parkinson's disease, Alzheimer's disease, and Huntington’s disease.

26. The method of claim 23, wherein the disorder comprises an infection by a pathogenic microorganism.

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