Lipid-mediated intracellular delivery of recombinant bioprotacs for rapid degradation of undruggable proteins

Abstract

The present disclosure provides recombinant fusion proteins comprising an E3 ubiquitin ligase domain, a small protein binding scaffold, and an anionic polypeptide. The present disclosure also provides compositions comprising the fusion proteins disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein. The small protein binding scaffold is designed to target an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain. The compositions disclosed herein are useful for treating cancers, inflammatory diseases, or neurodegenerative disorders.

Description

P-635623-PC LIPID-MEDIATED INTRACELLULAR DELIVERY OF RECOMBINANT BIOPROTACS FOR RAPID DEGRADATION OF UNDRUGGABLE PROTEINS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT.

[0001] This invention was made with government support under grant CA241661 awarded byNational Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] The present disclosure relates in general to the field of proteolysis targeting chimeras (PROTACs). In one embodiment, the present disclosure provides compositions and methods for cytosolic delivery of recombinant biological PROTACs. BACKGROUND OF THE INVENTION

[0003] Many endogenous proteins have smooth surfaces that preclude modulation by conventional drugs. The inability to develop small-molecule binders against these “undruggable” proteins remains a major hurdle in the biopharmaceutical industry and limits therapeutics development for many diseases. Crucially, targeted inhibition of oncogenic drivers is a valuable treatment strategy in many cancers, as key molecular underpinnings leading to tumor initiation, maintenance, and metastasis have been elucidated. Despite decades of research documenting the roles of aberrant proto-oncogenes in driving tumor growth, the current arsenal of FDA-approved inhibitors against high-profile targets including Ras, Myc and p53 is lacking. Even when successful molecules progress to market, their therapeutic scope is limited as highlighted by two recently-developed KRASG12Cinhibitors: sotorasib and adagrasib. While the discovery of these first KRAS inhibitors marks an important milestone in medicinal chemistry, these drugs can only target a subset of Ras variants found in a fraction of cancers. Effective inhibitors of Ras and other high-priority targets are highly desirable, but discovery of lead compounds remains challenging.

[0004] Efforts to overcome intractable proteins have looked beyond direct inhibition. One such approach is small-interfering RNA (siRNA), 21-25 nucleotide oligos that silence protein expression at the transcript level. While siRNA can target any protein-coding mRNA, they come with drawbacks. Critically, the efficiency of siRNA-mediated knockdown depends on the target protein’s intrinsic turnover rate, and depletion of proteins with long half-lives can lag or evenP-635623-PC outlast mRNA degradation. Non-specific protein knockdown is also possible through imperfect complementarity with off-target mRNA, leading to potential safety concerns. To address these limitations, a fully post-translational degradation system is needed.

[0005] Recently, proteolysis targeting chimeras (PROTACs) have gained considerable attention as a drug class for their ability to degrade proteins catalytically. PROTACs are heterobifunctional molecules that simultaneously engage endogenous ubiquitin-proteasome system (UPS) machinery and proteins of interest (POI) to induce POI degradation. Structurally, PROTACs comprise three domains: a POI-binding warhead, an E3-recruiting ligand, and a chemical linker separating the two binding moieties. Since they work by inducing proximity between E3s and POIs, PROTACs can be developed from any small-molecule binder, whereas traditional inhibitors require binding in active or allosteric sites to exert their pharmacological effects. The promise of targeted protein degradation has ignited interest in PROTACs, and in the past decade, dozens of oncology-focused degraders have entered clinical trials. Although they do not require binding to POI functional sites for activity, PROTACs still need high-affinity warheads for target engagement. Such ligands are difficult — if not impossible — to develop for intrinsically disordered proteins and proteins lacking hydrophobic pockets. Additionally, successful degradation necessitates the assembly of stable POI:PROTAC:E3 ternary complexes, but productive complex formation is dictated by complex interactions at the POI:E3 interface and are difficult to predict a priori. Thus, screening of both linker composition and linker length is often needed to empirically optimize degrader activity. This screening process is time-consuming and can oftentimes be unfruitful in generating active degraders.

[0006] In a related approach, fusion of a protein-based binder to either an E3 ligase or an E3 adapter results in a recombinant “bioPROTAC”. Also known as ubiquibodies or AdPROMs, these biologics selectively ubiquitinate target proteins for UPS-mediated degradation. Unlike their small-molecule counterparts, bioPROTAC warheads are directly fused to E3 domains and do not depend on the recruitment of endogenous ligases. This design has demonstrated remarkable versatility being modular with respect to both the warhead and the E3 ligase. In one example, Lim et al. produced bioPROTACs that successfully degraded the same substrate with four different binding scaffolds and seven different E3 ligase adapters. Importantly, bioPROTAC substrate specificity is conferred by protein scaffolds rather than small-molecule ligands. Since they operate via protein-protein interactions (PPI) spanning large contact areas, bioPROTACs can degrade targets completely inaccessible to small-molecule ligands. Also, the collection of targetableP-635623-PC proteins is vast, benefiting from the tremendous wealth of available binding proteins. To date, numerous scaffolds including nanobodies, designed ankyrin repeat proteins (DARPins), monobodies, and affibodies have been extensively developed for exquisite specificity and nanomolar-to-picomolar affinity. Taken together, bioPROTACs can be developed against any protein, greatly expanding the degradation toolbox against undruggable targets to address unmet medical needs in oncology and other therapeutic areas.

[0007] To realize the full potential of bioPROTACs, methods to deliver these macromolecules into cells are urgently needed. Currently, intracellular delivery of recombinant bioPROTACs is typically achieved by electroporation or microinjection, but these physical techniques are low- throughput, cytotoxic, and unsuitable for clinical translation. Alternatively, DNA or mRNA encoding desired bioPROTACs can be transfected into cells for expression, but nucleic acid methods also face several challenges. Firstly, nucleic acids require multi-step processing including transcription, translation, and polypeptide folding prior to target degradation. In addition, stability is a concern, since mRNA molecules are highly susceptible to environmental nucleases, requiring controlled, RNase-free facilities and ultracold storage conditions to avoid degradation. Finally, some therapeutically-interesting bioPROTACs have been reported to express poorly as mammalian transgenes despite facile production in bacterial cultures. By contrast, cytosolic protein delivery is a direct method to introduce bioactive molecules into cells for immediate target degradation. Recombinant bioPROTACs offer distinct manufacturing and storage advantages, and in some cases, protein delivery may be the only option for difficult-to-express degraders.

[0008] Common approaches for cytosolic protein delivery include cell penetrating peptides (CPPs), virus-like particles, inorganic nanoparticles, and supramolecular polymer assemblies. However, these methods suffer from complex synthesis methods, endosomal entrapment, or potential toxicities arising from bioaccumulation of nanocarrier materials.

[0009] Thus, there is a need to develop improved compositions and methods for cytosolic delivery of recombinant bioPROTACs. SUMMARY OF THE INVENTION

[0010] In one embodiment, the present disclosure describes a drug-like bioPROTAC format that can be delivered into unmodified cells for the on-demand degradation of endogenous proteins. To accomplish this goal, experiments were first performed to examine E3 domains that induced potent degradation when fused to the N-terminus of small protein binding scaffolds. In one embodiment,P-635623-PC DARPins were chosen as model binding domains owing to their excellent thermostability and high-yield expression in E. coli cultures. bioPROTAC- and target-encoding plasmids were transfected into HEK 293T (293T) cells to identify lead candidates. Once top E3-DARPin formatted bioPROTACs were identified, their ability to interact with charged lipids was explored for cytosolic delivery. To validate degradation activity of purified proteins, off-the-shelf cationic Lipofectamine reagent was used to deliver Ras-targeting bioPROTACs into GFP-KRAS expressing reporter cells. After confirming bioactivity, an LNP library was screened to identify combinations of ionizable lipids and excipients for enhanced bioPROTAC transfection and robust degradation (Figure 1). The degradation dose-dependence and kinetics of the lead formulation were characterized and compared to those of bioPROTAC-encoding mRNA. The broad applicability of the present LNP-delivered bioPROTAC platform to degrade diverse proteins and proteins localized to various subcellular compartments was examined. Finally, it was shown that Ras-targeting bioPROTACs can inhibit the proliferation of a KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell line. The results presented herein expand the functionality of existing protein binders into shelf-stable, on-demand intracellular degraders.

[0011] In one embodiment, the present disclosure provides a recombinant fusion protein comprising an E3 ubiquitin ligase domain, a small protein binding scaffold, and an anionic polypeptide, wherein the small protein binding scaffold targets an intracellular protein that is targeted to be degraded intracellularly.

[0012] In another embodiment, the present disclosure provides a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein. In one embodiment, the cationic agent is an ionizable lipid-like nanoparticle (LNP).

[0013] In another embodiment, the present disclosure provides a method of targeted intracellular degradation of a target protein, comprising contacting a cell with a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein, wherein the small protein binding scaffold of the fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of the fusion protein.

[0014] In another embodiment, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein, wherein the small protein binding scaffold of the fusion proteinP-635623-PC targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of the fusion protein.

[0015] These and other aspects of the invention will be appreciated from the ensuing descriptions of the figures and detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0017] Figure 1 shows one embodiment of lipid-mediated exogenous bioPROTAC delivery system. Purified bioPROTACs include an E3 ligase, binding domain, ApP, and GFP s11 tag. The fusion proteins are formulated as LNPs including ionizable / cationic lipids, neutral helper lipids, and lipid-anchored PEG. LNP:bioPROTAC can be delivered intracellularly, and upon endosomal escape, bind to and degrade target proteins. Cytosolic protein delivery is verified by s11 complementation with GFP(1-10) expressed in reporter cell lines.

[0018] Figures 2A-2J show screening of E3 domains for bioPROTAC development. Figure 2A shows protein designs for GFP-directed degradation. Figure 2B shows experimental design of co- transfection assays in 293T cells. 3G124 can bind to GFP-KRAS via the GFP handle and polyubiquitinate the target, marking it proteasomal destruction. Figures 2C-2H show flow cytometry results of 293T cells 48-hours after co-transfection with degrader- and target-encoding plasmids. Data are normalized geometric mean fluorescence intensity. No degradation was observed with the binder control (3G124) or CHIP-3G124. Both SKP2 and SOCS2 induced modest dose-dependent degradation. SPOP and IpaH9.8 transfection resulted in a dramatic reduction of target fluorescence. Figure 2I shows Western blot analysis of 293T lysates following co-transfection of 2.0µg degraders (except IpaH9.8) and 0.5µg of GFP-KRAS. Figure 2JI shows Western blot analysis of 293T lysates following co-transfection of 0.5µg GFP-KRAS and varying amounts of IpaH9.8-3G124.

[0019] Figures 3A-3G show identification of lead E3 for bioPROTAC development. Figure 3A shows either IpaH9.8 or SPOP was cloned at the N-terminus of Ras-binding K27. Figure 3B shows proposed mechanism of GFP-KRAS degradation via binding of the KRAS handle. Figure 3CP-635623-PC shows representative flow cytometry histograms 24 hours after co-transfection of the indicated bioPROTAC and GFP-KRAS. Either 2.0µg of SPOP-K27 / K27n3 plasmid or 0.25µg of IpaH9.8- K27 / K27n3 plasmid were co-transfected along with 0.5µg of GFP-KRAS plasmid. Figure 3D shows normalization and quantitation of the results of Figure 3C. Data shown are mean ± SD of n = 3 separately transfected wells. A one-sample, two-tailed t test was performed. ** p ≤ 0.01, *** p ≤ 0.001. Figure 3E shows design of full bioPROTAC and negative controls. Figure 3F shows schematic of purified protein transfection using cationic Lipofectamine 2000. Figure 3G shows flow cytometry histograms of 293T GFP-KRAS cells 8 hours after Lipofectamine transfection with either purified IpaH9.8-K27 or purified SPOP-K27 bioPROTACs.

[0020] Figures 4A-4F show characterization of IpaH9.8-based bioPROTAC delivery and degradation with Lipofectamine transfection. Figure 4A shows degradation efficiency of bioPROTAC and various controls in 293T GFP-KRAS stable cells complexed with Lipofectamine or incubated as naked proteins. Figure 4B shows flow cytometry analysis of 293T GFP(1-10) cells for GFP-positive population following treatment with the same conditions as Figure 4A. The dotted line represents the 1% threshold used to gate GFP-positive cells. Figure 4C shows fold- change in MFI quantified by flow cytometry with the same treatment conditions as Figures 4A and 4B. The dotted line represents the baseline GFP levels normalized to 1. Figure 4D shows the dose- dependence of degradation efficiency on IpaH9.8-K27 transfection amount. For each protein dose, 2µL Lipofectamine 2000 was used for complexation, and cells were analyzed 8 hours post- delivery. Figure 4E shows GFP-positive 293T GFP(1-10) cells following incubation with Lipofectamine:bioPROTAC. Figure 4F shows fold-change MFI of 293T GFP(1-10) cells following incubation with Lipofectamine:bioPROTAC. For each protein dose, 2µL Lipofectamine 2000 was used for complexation, and cells were analyzed 8 hours post-delivery Data are mean ± SD of n = 3 separately transfected wells. For Figures 4A-4C, two-way ANOVA was performed followed by multiple comparisons testing. ns p > 0.05, *** p ≤ 0.001, **** p < 0.0001.

[0021] Figures5A-5I show cytosolic bioPROTAC delivery by LNPs. Figure 5A shows microfluidic mixing of an aqueous bioPROTAC solution with an ethanol solution of lipids, PEG and cholesterol was used to formulate protein LNPs. Figure 5B shows flow histograms of 293T GFP-KRAS cells treated with bioPROTACs either complexed with Lipofectamine 2000 or formulated as LNPs. Figure 5C shows quantitation of Figure 5B. Figure 5D shows dose- dependent degradation in 293T GFP-KRAS with LNPs encapsulating either an active bioPROTAC or a non-binding control. Figure 5E shows acute cytotoxicity of LNPs in 293T cellsP-635623-PC was determined by an LDH assay following treatment with the K1 formulation. Figure 5F shows GFP-positive 293T GFP(1-10) cells following treatment with K1:bioPROTAC. Figure 5G shows fold-change MFI of 293T GFP(1-10) cells following treatment with K1:bioPROTAC. Figure 5H shows representative Western blots of 293T lysates after cells were treated with either bioPROTAC protein only or LNPs encapsulating Ras binders, Ras degraders (bioPROTAC), or control (bioPROTAC null). Figure 5I shows quantitation of western blot degradation by band densitometry normalized to an untreated control (dotted line). For all delivery experiments, cells were incubated with proteins or LNPs for 8 hours prior to analysis. Data for Figure 5D, 5F, and 5G are mean ± SD of n = 3 separately delivered wells. Data for Figure 5E and 5I are mean ± SD of n = 4. For Figure 5I, one-way ANOVA followed by multiple comparisons was performed. * p ≤ 0.05, ** p ≤ 0.01, **** p < 0.0001.

[0022] Figures 6A-6G show degradation kinetics of K1 formulation of IpaH9.8-based bioPROTACs. Figure 6A shows dual reporting 293T GFP-KRAS / iRFP-CaaX cells were left untreated and monitored for 12 hours by fluorescence microscopy. Figure 6B shows representative fluorescent images of reporter cells treated with the K1 LNP formulation with bioPROTAC protein as cargo. Figure 6C shows representative fluorescent images of reporter cells treated with an LNP formulation of bioPROTAC-encoding mRNA. Figure 6D shows fluorescence intensity of individual cells from Figure 6A. Figure 6E shows fluorescence intensity of individual cells from Figure 6B. Figure 6F shows fluorescence intensity of individual cells from Figure 6C. Between 400-500 single cells were analyzed at each time point over the 12-hour treatment window, and the mean is represented. Figure 6G shows the duration of bioPROTAC-mediated degradation was determined by flow cytometry. Scale bar applies to all microscopy images in Figures 6A-6C and is equal to 20µm.

[0023] Figures 1A-7F show global profiling of 293T proteome following Ras bioPROTAC treatment. Volcano plots display proteins identified from tandem mass spectrometry following 8- hour treatment with either Figure 7A shows K1-delivered Ras bioPROTACprotein, Figure 7B shows LNP-delivered bioPROTACmRNA.Figure 7C shows K1-delivered null bioPROTACprotein. Upregulated and downregulated proteins are indicated as red and blue data points respectively. Both NRAS and KRAS were identified in all conditions and highlighted in volcano plots. Figure 7D shows Venn diagram quantifying downregulated proteins from all treatment groups. The 8 downregulated proteins shared between both active bioPROTAC (protein and mRNA delivery) groups are shown. Figure 7E shows GO biological process enrichment analysis was performed onP-635623-PC downregulated proteins unique to mRNA treatment, and the top 10 terms by adjusted p-value were returned. Figure 7F shows the log2fc for all GTPases identified as significantly downregulated in either mRNA or protein bioPROTAC treatment are plotted. Proteins were classified based on homology to Ras-family GTPases. The dotted line marks the log2fc cutoff -1, used for identification of differentially downregulated proteins. For all treatment groups, n = 1. Log2 fold- change ratios were calculated against an untreated control group.

[0024] Figures 8A-8E show LNP-delivered bioPROTACs are modular and widely-active. Figure 8A shows a schematic for “plug-and-play” design of final bioPROTAC format. Figure 8B shows four DARPins targeting three different proteins were cloned into the bioPROTAC template. Figure 8C shows Western blot analysis reveals degradation of endogenous Jnk by K1:J1 / 2_2_25 bioPROTAC in 293T and degradation of Erk by K1:EpE89 bioPROTAC. No effect on Bcl-xL was observed following incubation with K1:012_F12 bioPROTAC. Figure 8D shows the anti- GFP bioPROTAC IpaH9.8-3G124-D25-s11 was formulated as K1 LNPs and delivered to HeLa cells stably expressing GFP-fusion proteins localized to the mitochondria, cytosol, and nucleus. Degradation was analyzed by fluorescence microscopy following treatment with 100nM protein for 8 hours. Figure 8E shows flow cytometric analysis was performed on HeLa and U2OS cells expressing various GFP-fusion proteins following treatment with 100nM GFP bioPROTAC (K1 LNP, orange trace). To demonstrate target specificity, Ras-targeting bioPROTACs were included as a control (red trace). Scale bar applies to all microscopy images in Figure 8D and is equal to 50µm.

[0025] Figures 9A-9I show inhibition of proliferation of Ras-dependent pancreatic cancer cells. Figures 9A-9G show Ras and pErk band densitometry results from MIA PaCa-2 lysates following treatment with K1:IpaH9.8-K27n3-D25-s11 protein (Figure 9A), K1:IpaH9.8-K27-D25-s11 protein (Figure 9B), C12-200:mRNA encoding the Ras bioPROTAC (Figure 9C) K1:IpaH9.8C337A-K27-D25-s11 protein (Fig. 9F), or K1:IpaH9.8C337A-K27n3-D25-s11 protein (Fig. 9G). Cells were treated at various doses for 8 hours before lysis and Western blot analysis. Figure 9D shows cell proliferation was assayed with the xCELLigence real-time cell analysis (RTCA) system, and normalized growth was calculated at 24 hours post-treatment with bioPROTAC proteins either on their own or formulated as K1 LNPs. Data were normalized to untreated cells (dotted line). Figure 9E shows MIA PaCa-2 cells were treated with C12-200 LNPs encapsulating bioPROTAC mRNA, proliferation was assessed by xCELLigence RTCA, and the 24-hour growth was calculated. All data were normalized to untreated controls. For Figure 9D,P-635623-PC data in protein-only conditions are mean ± SD of n = 4 wells across two independent experiments. For K1:bioPROTAC treatment conditions, data are mean ± SD of n = 6 wells across three independent experiments. For Figure 9E, data are mean ± SD of n = 6 wells across two independent experiments. An ordinary two-way ANOVA test was performed followed by multiple comparisons testing. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01. Fig. 9C shows Ras and pErk band densitometry results from MIA PaCa-2 lysates following treatment with C12-200:mRNA encoding the IpaH9.8- K27 bioPROTAC. Fig.9H shows cell proliferation was assayed with the xCELLigence real-time cell analysis (RTCA) system, and normalized growth was calculated at 24 hours post-treatment with bioPROTAC proteins formulated as K1 LNPs (56nM dose). Data are the mean ± SD of either n = 4 (IpaH9.8C337Avariants) or n = 7 (IpaH9.8 WT controls) biological replicates. Fig. 9I shows MIA PaCa-2 cells were treated with C12-200 LNPs encapsulating bioPROTAC mRNA, proliferation was assessed by xCELLigence RTCA, and the 24-hour growth was calculated. All data were normalized to untreated controls. Band densitometry was performed at 8 hours post- treatment, and n = 1 biological replicate for each data point. A two-way ANOVA test was performed followed by multiple comparisons testing. ** p ≤ 0.01, *** p ≤ 0.001. For G, the experiment was performed twice, and the two biological replicates are shown. Source data are provided as a Source Data file.

[0026] Figures 10A-10F show characterization of GFP-targeting bioPROTACs. Figure 10A shows binding of 3G124 to GFP with or without ApP fusion. Data are mean of n = 2 wells. Figure 10B shows SDS-PAGE confirms that purified GFP bioPROTACs run at their predicted molecular weights. Figure 10C shows GFP and SPOP-3G124-D25-s11 incubated together and complexes were analyzed by analytical SEC to evaluate binding in solution. Figure 10D shows SDS-PAGE of primary peaks from Figure 10C show co-elution of GFP with SPOP-based bioPROTAC. Figure 10E shows chromatogram of GFP and IpaH9.8-3G124-D25-s11 complexes following incubation and SEC. Figure 10F shows SDS-PAGE of primary peaks from Figure 10E show co-elution of GFP with IpaH9.8-based bioPROTAC. For Figure 10C-10F, the red box outlines bioPROTAC- containing fractions, while the green box indicates GFP-only fractions. Numbers above peaks and gel images in Figure 10C-10F correspond to the elution fraction number.

[0027] Figures 11A-11H show characterization of Ras-binding bioPROTACs. Figure 11A shows PyMol model of IpaH9.8-K27 bioPROTAC binding to KRAS. The catalytic cysteine residue responsible for target ubiquitination is highlighted in cyan. Figure 11B shows SDS-PAGE was performed to verify the successful purification of Ras-targeting bioPROTACs and control proteins.P-635623-PC Figures 11C-11H show normalized chromatograms of SPOP- and IpaH9.8-fusion proteins. SPOP-fusions exhibit larger retention volumes compared to IpaH9.8 chimeras despite a lower monomeric molecular weight, indicating oligomerization.

[0028] Figures 12A-12B show binding of purified Ras-targeting bioPROTACs to KRAS. Figure 12A shows a schematic of DARPinK27 with minimal modifications compared to the complete bioPROTAC with an N-terminal IpaH9.8 domain and a C-terminal D25 ApP. Figure 12B shows results of binding assay that show the affinity of the complete anti-Ras bioPROTAC was found to be minimally affected by fusion to an E3 domain and a charged peptide sequence. Data are mean ± SD of n = 3 wells.

[0029] Figure 13 shows in vitro ubiquitination assay. Purified bioPROTACs containing a binding domain (K27) or non-binding control (K27n3) were incubated with KRAS, E1, E2, and ubiquitin for 2 hours, and samples were analyzed by western blotting. Molecular weight bands above 24 kDa (recombinant KRAS) indicate ubiquitination (Ub) of KRAS.

[0030] Figures 14A-14E show comparison of bioPROTAC- and siRNA-mediated degradation rates. Figure 14A shows Ras-targeting bioPROTAC (500nM) was complexed with Lipofectamine 2000 and delivered into 293T GFP-KRAS cells. Figure 14B shows GFP-positive 293T GFP(1- 10) cells following treatment with Lipofectamine:bioPROTAC. Figure 14C shows fold-change MFI of 293T GFP(1-10) cells following treatment with Lipofectamine:bioPROTAC. Figure 14D shows degradation in 293T GFP-KRAS cells transfected with a negative control siRNA or two anti-KRAS siRNA. Figure 14E shows flow cytometry was performed up to 12 hours post- transfections and the degradation rate for each modality was estimated using a first-order decay equation. Data are mean ± SD of n = 3 separately transfected wells.

[0031] Figure 15 shows stability of purified proteins. Purified IpaH9.8-K27-D25-s11 was stored at either 4°C or -80°C for 28 days prior to Lipofectamine complexation. For both storage conditions, degradation efficiency was no different compared to newly-purified bioPROTAC. Data are mean ± SD of n = 3 separately transfected wells.

[0032] Figures 16A-16E show characterization of LNP:bioPROTAC formulations. Figure 16A shows degradation efficiency of LNP library in 293T GFP-KRAS cells. Figure 16B shows degradation efficiency of K1 was measured over the course of 1 week and did not exhibit a decline in activity. Figure 16C shows Cryo-TEM micrograph of K1:bioPROTAC LNPs. Scale bar = 100nm. Figure 16D shows encapsulation efficiency of TAMRA-labeled bioPROTAC within K1 LNPs was determined by size exclusion column chromatography. Figure 16E shows fractionsP-635623-PC corresponding to the major peak in Figure 16D were pooled and analyzed by dynamic light scattering to confirm LNP content. Data for Figure 16A are mean ± SD of n = 3 measurements.

[0033] Figures 17A-17F show degradation of endogenous Ras in a panel of cancer cell lines. Figure 17A shows representative Western blot of A549 non-small cell lung cancer lysates after cells were treated with 100nM LNPs formulated either active bioPROTAC, a non-binding bioPROTAC, or an E3-deficient control. Figure 17B shows band densitometry was performed on n = 5 replicates of A549 cells with the dotted line representing the normalized intensity of the untreated control. Figure 17C shows experiments as in Figure 17A but with HCT116 colorectal cancer cells. Figure 17D shows band densitometry was performed on n = 5 replicates of HCT116 cells. Figure 17E shows experiments as in Figure 17A but with HT1080 fibrosarcoma cells. Figure 17F shows band densitometry was performed on n = 4 replicates of HT1080 cells. Data mean ± SD. One-way ANOVA followed by multiple comparisons was performed. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

[0034] Figure 18 shows dose-dependent degradation in 293T GFP-KRAS cells treated with LNP:mRNA encoding the Ras-targeting bioPROTAC. Cells were treated for 8 hours and analyzed by flow cytometry. Data are mean of n = 2 wells.

[0035] Figures 19A-19D show controls for degradation kinetics study. Figure 19A shows dual reporting 293T GFP-KRAS / iRFP-CaaX cells were treated with K1:bioPROTAC(null) and monitored for 12 hours by fluorescence microscopy. Figure 19B shows single-cell analysis of 400-500 cells at each time point following delivery of null bioPROTAC. Figure 19C shows representative fluorescent images of reporter cells treated with the B6:K27-D25-s11. Figure 19D shows single-cell analysis of 400-500 cells at each time point following delivery of K27-D25-s11. Scale bar applies to all microscopy images in Figures 19A and 19C and is equal to 20µm.

[0036] Figures 20A-20C show additional proteomics sample analyses. Figure 20A shows immunoblotting of 293T extracts following protein or mRNA bioPROTAC treatment confirms expected Ras knockdown prior to MS analysis. Protein delivery samples were treated in duplicate. Figure 20B shows GO enrichment analysis was performed on the downregulated proteins shared between LNP:bioPROTACmRNA and K1:bioPROTAC (null)protein treatment groups, and the top 10 terms by adjusted p-value were returned. Figure 20C shows STRING DB interaction network for U2AF and related proteins associated with RNA splicing identified from pathway enrichment analysis.

[0037] Figures 21A-21E show purification and characterization of a catalytically-deadP-635623-PC bioPROTAC. Figure 21A shows size exclusion chromatography chromatograms of Ras-targeting bioPROTACs incorporating either the wild-type (WT) IpaH9.8 or an IpaH9.8 with its catalytic cysteine residue mutated to alanine (C337A). Figure 21B shows SDS-PAGE analysis of WT or C337A bioPROTACs under non-reducing (NR) or reducing (R) conditions. Figure 21C shows in vitro KRAS ubiquitination assay using WT or C337A bioPROTACs. Figure 21D shows KRAS binding assays were performed with active, catalytically-dead, or non-binding bioPROTACs. Each data point is the mean of n = 2 wells. Figure 21E shows autoubiquitination was assessed in vitro with TAMRA-labeled IpaH9.8WTand IpaH9.8C337AbioPROTACs.

[0038] Figures 22A-22G show mechanistic interrogation of Ras bioPROTAC activity. Figure 22A shows the degradation activity of K1:bioPROTAC LNPs were assayed by flow cytometry in 293T GFP-KRAS cells. Either WT or C337A IpaH9.8 bioPROTACs were formulated as K1 LNPs and delivered into cells. LNP-treated cells were additionally incubated with or without MG-132. (n=3 technical replicates). Figure 22B shows delivery efficiency represented as percent split GFP complementation in 293T GFP(1-10) cells treated with K1 LNPs encapsulating either active or catalytically-dead bioPROTACs (n=3 technical replicates). Figure 22C shows the fold-change MFI in 293T GFP(1-10) cells following treatment with K1:bioPROTAC. Figure 22D shows representative Western blot of endogenous Ras in 293T extracts following K1:bioPROTAC treatment with or without MG-132. Figure 22E shows quantitation of samples represented in Figure 22D (n = 3 Western blots for the conditions outlined in Figure 22D). Data were normalized to untreated cells without MG-132 (dotted line). Data are mean ± SD of n = 3 biological replicates. Treatment time for all panels was 8 hours. Two-way ANOVA followed by multiple comparisons testing was performed. ns p > 0.05, ** p = 0.006, **** p < 0.0001. Figure 22F shows changes to cytosolic delivery in 293T GFP(1-10) cells (x-axis) and target degradation in 293T GFP-KRAS cells (y-axis) following K1:bioPROTAC (IpaH9.8WT-K27-D25-s11) treatment in combination with proteasomal and / or lysosomal inhibitors (10µM MG-132, 50µM chloroquine, 100nM bafilomycin A1). Figure 22G shows endogenous Ras levels in 293T cell lysates following treatment with K1:bioPROTAC with or without proteasomal / lysosomal inhibitors. Treatment time for all panels was 8 hours, and 100nM bioPROTAC was used in protein delivery groups. Source data are provided as a Source Data file.

[0039] Figures 23A-23D show Western blot analysis of endogenous Ras degradation in a pancreatic cancer cell line. Figure 23A shows dose-dependent effects of various LNP cargo on endogenous Ras, pErk, and total Erk levels following 8-hour treatment and epidermal growthP-635623-PC factor stimulation. Figure 23B shows time-course of Ras, pErk, and total Erk levels in MIA PaCa- 2 cells treated with degrader-encapsulating LNPs. For protein delivery, the K1 formulation was used, and a dose of 100nM was chosen. For mRNA delivery, C12-200 LNPs were formulated, and a dose of 150ng / mL was chosen. Figs. 23C-23D show Western blot analysis of endogenous protein levels in the MIA PaCa-2 pancreatic cancer cell line following degrader treatment. Fig. 23C shows dose- dependent effects of various LNP cargo on endogenous Ras, pErk, and total Erk levels in MIA PaCa-2 following 8- hour treatment and epidermal growth factor stimulation. Fig.23D shows time-course of Ras, pErk, and total Erk levels in MIA PaCa-2 cells treated with degrader-encapsulating LNPs. For protein delivery, the K1 formulation was used, and a dose of 100nM was chosen. For mRNA delivery, C12-200 LNPs were formulated, and a dose of 150ng / mL was chosen. Source data are provided as a Source Data file.

[0040] Figures 24A-24J show screening of ionizable lipids for Ras targeted therapy in pancreatic cancer and characterization of LNP degraders for Ras-targeted therapy in pancreatic cancer. Figure 24A shows 8 ionizable lipids were substituted for the C12-200 lipid used in the base K1 formulation. MIA PaCa-2 cells were treated with 100nM protein LNPs for 8 hours. Cells were stimulated with human epidermal growth factor (EGF) prior to lysis, and extracts were blotted for Ras, pErk and Erk levels. Figure 24B shows both normalized Ras and normalized pErk levels were quantified, and 4 lipids were chosen for further testing (red points). Figures 24C-24H show MIA PaCa-2 pancreatic cancer cells were treated with 100nM active and null bioPROTACs either as free protein or as LNP:protein formulations. In total, 4 lipids identified from screening were tested, and C12-200 (K1 formulation) was included as a benchmark. Cell proliferation was assayed using the xCELLigence real-time cell analysis system. Data were normalized to untreated controls. For protein-only experiments (Fig. 24C), data points represent the mean of n = 2 biological replicates. In panel of Fig.24D, low dose (blue and green) data points are the mean ± SEM of n = 7 biological replicates, and high dose (purple and red) are mean ± SEM of n = 6 biological replicates. For Figs.24E- 24H, data are mean ± SEM of n = 3 biological replicates. For each lipid, two-way ANOVA was performed followed by comparisons between active and null bioPROTACs at matched doses. Only statistically-significant results are indicated. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Fig. 24I shows Ras degradation was confirmed for each LNP:protein formulation (100nM) by Western blotting. Fig. 24J shows normalized proliferation of MIA PaCa-2 cells treated with C12-200 LNPs encapsulating Ras bioPROTAC mRNA. Data are mean of n = 2 biological replicates, with each biological replicate containing 3 technical replicates. Source dataP-635623-PC are provided as a Source Data file.

[0041] Figure 25 shows a representative gating strategy for flow cytometry experiments. For both degradation experiments and split GFP delivery assays, geometric mean GFP levels were calculated from the entire histogram. For split GFP delivery assays, an additional gate (GFP+ window) was applied, and all cells within this window were considered GFP-positive. This gate was made such that only 1% of the untreated GFP(1-10) cell sample would be considered GFP- positive. DETAILED DESCRIPTION OF THE INVENTION

[0042] In recent years, post-translational protein degradation has emerged as a powerful therapeutic modality. Relying on “event-driven” pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is still limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small- molecule PROTACs, bioPROTACs have higher success rates, are subject to fewer design constraints, and are more easily generated for selected targets. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. The present disclosure describes an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles (LNPs), these modified bioPROTACs can potently and rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. The bioPROTAC format disclosed herein can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. Using LNPs encapsulating Ras-targeting bioPROTACs as example, the present disclosure demonstrates therapeutic utility by inhibiting proliferation in a Ras-driven pancreatic cancer cell line. In summary, the present disclosure introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that have long eluded chemical inhibition.

[0043] The subject matter here may be understood more readily by reference to the following detailed description which forms part of this disclosure. It is to be understood that this inventionP-635623-PC is not limited to the specific products, methods, conditions or parameters described or shown here, and that the terminology used here is for the purpose of describing certain embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0044] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.

[0045] In this disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term “plurality”, as used herein, means more than one.

[0046] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0047] As used herein, the terms "component," "composition," "composition of compounds," "compound," "drug," "pharmacologically active agent," "active agent," "therapeutic," "therapy," "treatment," or "medicament" are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and / or physiologic effect by local and / or systemic action.

[0048] As used herein, the terms "treatment" or "therapy" (as well as different forms thereof) include preventative (e.g., prophylactic), curative or palliative treatment. As used herein, the term "treating" includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease, or disorder.

[0001] The terms "subject," "individual," and "patient" are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with theP-635623-PC pharmaceutical composition according to the present invention, is provided. The term "subject" as used herein refers to human and non-human animals. The terms "non-human animals" and "non- human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.

[0049] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0050] In the description presented herein, each of the steps of the invention and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present invention.

[0051] It is appreciated that certain features of the invention, 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 invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0052] In one embodiment, the present disclosure provides a recombinant fusion protein comprising (i) an E3 ubiquitin ligase domain, (ii) a small protein binding scaffold, and (iii) an anionic polypeptide, wherein the small protein binding scaffold targets an intracellular protein that is targeted to be degraded intracellularly. E3 Ubiquitin Ligases

[0053] Ubiquitination is a post-translational modification of proteins that involves the covalent attachment of one or more ubiquitins (Ub). It plays an important role not only in proteasomal degradation but also in many other processes such as protein-protein interactions, subcellular localization, and DNA repair. Ubiquitination requires the actions of a cascade of three enzymes:P-635623-PC E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. E1 activating enzymes transfer ubiquitin (Ub) in an ATP-dependent manner to the E2 conjugating enzyme. The E3 ubiquitin ligase bridges the E2 and the target protein (the substrate) to facilitate the transfer of Ub to a lysine or the N-terminus of the target.

[0054] The recombinant fusion proteins disclosed herein encompass all the E3 ubiquitin ligases known in the art. While humans only have two E1 activating enzymes, and about 30 different E2 conjugating enzymes, there are over 600 different E3 ligases. According to the differences of structure and function, E3 ligases can be divided into different types such as HECT (Homologous to E6AP C-terminus) type, U-box type, RING (Really Interesting New Gene) finger type, and RBR (RING-between-RING) type. Numerous E3 ligases belonging to each of these families are widely known in the art.

[0055] It is also known in the art that there is a new family of E3 ligases that is not found in eukaryotic cells and is known as the novel E3 ligase family. Members of this family include, but are not limited to, the IpaH proteins from Shigella flexneri, as well as SlrP, SspH1, and SspH2 from S. enterica serovar Typhimurium. Other E3 ligases belonging to the bacterial novel E3 ligase family are widely known in the art. Small Protein Binding Scaffold

[0056] Many biological functions depend on specific protein-protein interactions. A prototype of binding protein is an antibody, a highly selective and adaptive molecule capable of binding to a huge spectrum of partners. The antibody-based binders have been indispensable not only in research experiments but also in clinical trials. However, several suboptimal properties of these molecules such as their large size and cross-reactivity motivated the development of binders with alternative structures. These novel artificial high-affinity binders are called small non-antibody protein binding scaffolds. Different scaffolds offer specific advantages and disadvantages. Various types of small protein binding scaffolds are known in the art. Examples of small protein binding scaffolds include, but are not limited to, designed Ankyrin repeat protein (DARPin), nanobody, affibody, monobody, nanobody, knottin, affimer, Nanofitin, avimer, or a Centyrins.

[0057] Ankyrin repeats (ARs), one of the most common protein motifs found in nature, are 33 amino acid long sequences composed of a β-turn followed by two anti-parallel α-helices and a loop. Various numbers of these individual ARs stack together to form ankyrin repeat proteins which function as protein binders. Recognizing the potential of these natural Ankyrin repeatP-635623-PC proteins as alternative target-binding domains, libraries of artificial stacked ARs, called designed Ankyrin repeat proteins (DARPins) were developed to allow for the generation of repeat protein binders against a defined target of interest. Each DARPin in these libraries typically contains between 2 and 6 repeating units; 2–4 repeats containing both fixed (framework sites required for correct AR folding) and variable (randomized sites leading to a diversity of target-binding capacity within the library) amino acid positions sandwiched between nonvariable N-terminal and C- terminal capping repeats. Expression of these genetic DARPin libraries using ribosome or phage display systems allows for the selection of DARPins with the capacity to bind a defined target of interest as well as refine binding affinity for that target. Anionic Polypeptides

[0058] The term “anionic polypeptide” refers to a polypeptide that has an anionic or a negative charge at physiologic pH. The anionic polypeptide may include a plurality of negatively charged amino acid residues or unnatural amino acid residues. In some embodiments, the anionic polypeptide may include a plurality of “repeats” of negatively charged amino acid residues. For example, the number of repeats of the negatively charged amino acid residues range from about 2 to about 50, about 10 to about 40, about 20 to about 30, or about 25 to about 30. In some embodiments, at least 50% of residues in the anionic polypeptide are repeats of negatively charged amino acid residues.

[0059] Examples of negatively charged amino acid residues are well known in the art. In some embodiments, the negatively charged amino acid residue is aspartic acid. In some embodiments, the anionic polypeptide comprises a plurality of aspartic acid residues. In some embodiments, the negatively charged amino acid residue is glutamic acid. In some embodiments, the anionic polypeptide comprises a plurality of glutamic acid residues. In some embodiments, the negatively charged amino acid residue is an unnatural amino acid. For example, the anionic polypeptide comprises a plurality of negatively charged unnatural amino acid residues.

[0060] In some embodiments, the negatively charged amino acid is a glutamic acid, aspartic acid, or negatively charged unnatural amino acid. In some embodiments, the anionic polypeptide comprises a plurality of glutamic acid, aspartic acid, and negatively charged unnatural amino acid residues. Linkers

[0061] In one embodiment, the fusion protein disclosed herein comprises a linker between the E3P-635623-PC ubiquitin ligase domain and the small protein binding scaffold. In another embodiment, the fusion protein disclosed herein comprises a linker between the small protein binding scaffold and the anionic polypeptide. In another embodiment, the fusion protein disclosed herein comprises a linker between the E3 ubiquitin ligase domain and the small protein binding scaffold, and a linker between the small protein binding scaffold and the anionic polypeptide. Various linkers and techniques are known in the art for linking molecules.

[0062] The term “linker”, “linker sequence”, “spacer”, or grammatical equivalents thereof refer to a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a preferred configuration. A number of strategies may be used to covalently link molecules together. These include but are not limited to polypeptide linkages between N- and C-terminus of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents. In one embodiment, the linker is a peptide bond, generated by recombinant techniques or peptide synthesis. In another embodiment, the linker is a cysteine linker. In yet another embodiment, it is a multi-cysteine linker. Choosing a suitable linker for a specific case where two polypeptide chains are to be connected depends on various parameters, including but not limited to the nature of the two polypeptide chains (e.g., whether they naturally oligomerize), the distance between the N- and the C-termini to be connected if known, and / or the stability of the linker towards proteolysis and oxidation. Furthermore, the linker may contain amino acid residues that provide flexibility. In one embodiment, the linker peptide may include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. Suitable lengths for this purpose include at least one and not more than 30 amino acid residues. In one embodiment, the linker is from about 1 to 30 amino acids in length. In another embodiment, the linker is from about 1 to 15 amino acids in length. In addition, the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not interfere significantly with the activity of the polypeptide. Thus, the linker peptide on the whole should not exhibit a charge that would be inconsistent with the activity of the polypeptide, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the monomers that would seriously impede the binding of monomer domains.

[0063] In some embodiments, useful linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shakerP-635623-PC potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Suitable linkers may also be identified by screening databases of known three-dimensional structures for naturally occurring motifs that can bridge the gap between two polypeptide chains. In one embodiment, the linker is not immunogenic when administered in a human subject. Thus, linkers may be chosen such that they have low immunogenicity or are thought to have low immunogenicity. Another way of obtaining a suitable linker is by optimizing a simple linker, e.g., (Gly4Ser)n, through random mutagenesis. Alternatively, once a suitable polypeptide linker is defined, additional linker polypeptides can be created to select amino acids that more optimally interact with the domains being linked. Other types of linkers that may be used in the compositions and methods provided herein include artificial polypeptide linkers and inteins. In another embodiment, disulfide bonds are designed to link the two molecules. In another embodiment, linkers are chemical cross-linking agents. For example, a variety of bifunctional protein coupling agents may be used, including but not limited to N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0064] In another embodiment, chemical linkers may enable chelation of an isotope. For example, Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to an antibody.

[0065] In another embodiment, the linker may be cleavable. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al., 1992, Cancer Research 52: 127-131) may be used. Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. Cationic Agent That Facilitates Cytoplasmic Delivery

[0066] In another embodiment, the present disclosure provides a composition comprising the recombinant fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein. In one embodiment, the anionic polypeptide of the fusion proteinP-635623-PC interacts with the cationic agent to facilitate cytoplasmic delivery of the fusion protein.

[0067] In one embodiment, the cationic agent is a nano-carrier. In one embodiment, the cationic agent is an ionizable carrier. In certain embodiments, the ionizable carrier includes an ionizable- lipid, polymer, or combination thereof. In some embodiments, the ionizable carrier is an ionizable lipid-like nanoparticle.

[0068] In one embodiment, the cationic agent comprises a cationic lipid. The term “cationic lipid” refers to a lipid which has a cationic or positive charge at physiologic pH. Cationic lipids can take a variety of forms including, but not limited to, liposomes or micelles. Cationic lipids useful for certain aspects of this disclosure are known in the art, and generally comprise both polar and non- polar domains, and bind to polyanions. Cationic lipids have been used in the art to deliver molecules to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; 8,569,256; 8,691,750; 8,748,667; 8,758,810; 8,759,104; 8,771,728; Lewis et al. 1996. Proc. Natl. Acad. Sci. 93:3176; Hope et al. 1998. Molecular Membrane Biology, 15:1, each of which is incorporated by reference herein in its entirety).

[0069] Examples of cationic lipids include, but are not limited to, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE®(e.g., LIPOFECTAMINE®2000, LIPOFECT AMINE®3000, LIPOFECTAMINE®RNAiMAX, LIPOFECTAMINE®LTX, LIPOFECTAMINE®MessengerMAXTM), LIPOFECTAMINE®CRISPRMaxTMCas9 Transfection Reagent, Invivofectamine, SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, CA), and Eufectins (JBL, San Luis Obispo, CA). Exemplary cationic liposomes can be made from N-[l-(2,3-dioleoloxy)-propyl]- N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP), 3P-[N-(N’,N’- dimethylaminoethane)carbamoyl]cholesterol (DC-Choi), 2,3,-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 1,2- dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB).

[0070] In another embodiment, the cationic agent comprises a cationic polymer. The term “cationic polymer,” as used herein, refers to a polymer having a net positive charge. Cationic polymers are well known in the art, and include those described in Samal et al., Cationic polymers and their therapeutic potential. Chem Soc Rev. 2012 Nov 7;41(21):7147-94; in published U.S. patent applications US2014 / 0141487, US2014 / 0141094, US2014 / 0044793, US2014 / 0018404,P-635623-PC US2014 / 0005269, and US2013 / 0344117; and in U.S. Pat. Nos. 8,709,466; 8,728,526; 8,759,103; and 8,790,664; the entire contents of each are incorporated herein by reference. Exemplary cationic polymers include, but are not limited to, polyallylamine (PAH); polyethyleneimine (PEI); poly(L- lysine) (PLL); poly(L-arginine) (PLA); polyvinylamine homo- or copolymer; a poly(vinylbenzyl- tri-Ci-C4-alkylammonium salt); polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, In vivo-jetPEI, TransIT-QR, a polymer of an aliphatic or araliphatic dihalide and an aliphatic N,N,N’,N’-tetra-Ci-C4-alkyl-alkylenediamine; a poly(vinylpyridin) or poly(vinylpyridinium salt); a poly(N,N-diallyl-N,N-di-Ci-C4-alkyl-ammoniumhalide); a homo- or co-polymer of a quaternized di-Ci-C4-alkyl-aminoethyl acrylate or methacrylate; POLYQUAD™; and a polyaminoamide.

[0071] Examples of cationic lipids, lipid-like materials and cationic polymers are disclosed herein, and additional suitable lipids and lipid-like materials are known in the art (see, e.g., those described in Akinc et al., Nature Biotechnology 26, 561-569 (2008), the entire contents of which is incorporated herein by reference).

[0072] In one embodiment, the cationic agent is a nano-carrier. Any suitable nano-carrier known in the art can be used. In one embodiment, the cationic agent is an ionizable lipid-like nanoparticle. For example, the ionizable lipid comprises a polyamine core structure reacted with an alkyl epoxide.

[0073] In one embodiment, the cationic agent comprises lipid-like nanoparticles (LNPs). Any suitable LNPs known in the art can be used. In one embodiment, the LNPs comprise ionizable or cationic lipid, cholesterol, neutral lipid, and PEG-lipid. The portions of these various components in an LNP composition can be formulated by one of ordinary skill in the art according to generally known methods and techniques.

[0074] In some embodiments, the nano-carrier is pegylated or coated with a material that increases solubility, increases biocompatibility, reduces opsonization, and / or extends circulation time. The nanoparticle may be further modified with a targeting ligand that confers specificity for a cell surface receptor. Methods of Uses

[0075] In one embodiment, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmicP-635623-PC delivery of the fusion protein, wherein the small protein binding scaffold of the fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of the fusion protein.

[0076] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0077] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0078] In one embodiment, "treating" refers to either therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder. Thus, in one embodiment, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in one embodiment, "treating" refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In one embodiment, "preventing" refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment, "suppressing" or "inhibiting", refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0079] The terms "subject," "individual," and "patient" are used interchangeably herein, and refer to human or non-human animals to whom treatment with a composition or formulation in accordance with the present disclosure is provided. The terms "non-human animals" and "non- human mammals" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (e.g. higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig,P-635623-PC goat, pig, cat, rabbits, cows, horses, or non-mammals such as reptiles, amphibians, chickens, and turkeys. In one embodiment, the mammal to be treated is human. The human can be any human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child. The human can be male, female, pregnant, middle-aged, adolescent, or elderly.

[0080] Pharmaceutical compositions suitable for use in the methods disclosed herein include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In one embodiment, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0081] In one embodiment, for any preparation used in the methods disclosed herein, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. In another embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0082] In one embodiment, the present disclosure provides a recombinant fusion protein comprising an E3 ubiquitin ligase domain, a small protein binding scaffold, and an anionic polypeptide, wherein the small protein binding scaffold targets an intracellular protein that is targeted to be degraded intracellularly.

[0083] In one embodiment, the E3 ligase domain comprises a mammalian E3 ligase, an E3 ligase adapter, or bacterial novel E3 ligase. As it is generally known in the art, the E3 ligase adapter in multi-unit E3 ligases, typically from the Cullin-RING family, is the subunit protein that serves to bridge the substrate to the rest of the E3 ligase complex. E3 ligase adapter comprises one domain that recognizes a substrate to be degraded and a conserved domain that recognizes a family ofP-635623-PC cullin RING ligases. Here, an E3-recruiting portion of the E3 ligase adapter proteins is used for bioPROTAC construction by replacing the native substrate binding domain with an alternate target-specific scaffold such as DARPin. RING E3 ligases that do not require a separate adapter also exist. Examples of the E3 ligase adapter include, but are not limited to, SPOP, VHL, SOCS2 and SKP2.

[0084] In one embodiment, the mammalian E3 ligase is a Really Interesting New Gene (RING) E3 ligase, Homologous to E6AP C-terminus (HECT) E3 ligase, a RING-between-RING (RBR) E3 ligase, or an U-box E3 ligase. Examples of the RING E3 ligase include, but are not limited to, COP1, Mdm2, and TRAF6. Examples of the HECT E3 ligase include, but are not limited to, E6AP, HUWEI, and HERC. Examples of the RBR E3 ligase include, but are not limited to, HHARI and UBAC. An example of the U-box E3 ligase is CHIP. Examples of the bacterial novel E3 ligase include, but are not limited to, SlrP, SspH1, SspH2, SspH3, IpaH1.4, IpaH 2.5, IpaH 4.5, IpaH 7.8, IpaH 9.8, IpaH a, IpaH b, IpaH c, IpaH d, IpaH e, NopM, RipAR, RipAW, RipV1, and RipV2.

[0085] In one embodiment, the small protein binding scaffold of the fusion protein is a designed Ankyrin repeat protein (DARPin), a nanobody, an affibody, a monobody, a nanobody, a knottin, an affimer, a Nanofitin, an avimer, or a Centyrins. Examples of the DARPin include, but are not limited to, DARPin 3G124, DARPin K27, DARPin K19, DARPin K13, DARPin EpE89, DARPin J1 / 2_2_3, or DARPin J1 / 2_2_25.

[0086] In one embodiment, the anionic polypeptide of the fusion protein comprises at least 50% negatively charged amino acids. In one embodiment, the anionic polypeptide comprises aspartic acid, glutamic acid, or a combination thereof. In one embodiment, the anionic polypeptide comprises about 10-50 aspartic acid exclusively. For example, the anionic polypeptide comprises about 10, 15, 20, 25, 30, 35, 40, 45 or 50 aspartic acid. In another embodiment, the anionic polypeptide comprises about 10-50 glutamic acid exclusively. For example, the anionic polypeptide comprises about 10, 15, 20, 25, 30, 35, 40, 45 or 50 glutamic acid. In another embodiment, the anionic polypeptide comprises about 10-50 amino acids, wherein these 10-50 amino acids comprise any combination or permutation of aspartic acid and glutamic acid. Examples of combinations of aspartic acid and glutamic acid include, but are not limited to, DDDEDDEDEEDEDEEDEDDDDEDEDEDDEDDEEEDDDEDD (ApP40), and DEDEEDEDEEDEDDDEEDEDEDDEDDEEED (ApP30).

[0087] In one embodiment, the intracellular proteins that can be targeted by the small protein binding scaffold of the present fusion protein include, but are not limited to, KRAS, HRAS, NRAS,P-635623-PC p53, Myc, NF-κB, β-catenin, estrogen receptor, and androgen receptor.

[0088] In an embodiment, the intracellular protein that is targeted to be degraded intracellularly (also called herein “intracellular protein”) is a Ras protein, a MAPK protein, a p53 protein, a Myc protein, a NF-κB protein, β-catenin, a Tau protein, an amyloid beta (Aβ) protein, estrogen receptor, androgen receptor, multidrug resistance proteins (MRP), B-cell lymphoma-2 (BCL2) family, Cyclin D1 protein, AKT protein, Mdm2 protein, Surviving protein, E2F, HIF-1alpha, STAT family, TNF, interleukin family, NPPB, NPPA, C-reactive protein, TGFbeta, alpha-synuclein, and / or mucin.

[0089] In certain embodiments the Ras protein selected from the group consisting of KRAS, HRAS and NRAS; the MAPK protein is selected from the group consisting of Erk and pErk; the Myc protein selected from the group consisting of c-myc (MYC), I-myc (MYCL) and n-myc (MYCN); and the NF-κB protein selected from the group consisting of NF- κB1, NF- κB2, relA, RelB and c-Rel.

[0090] Transcription factor NF-κB regulates many aspects of innate and adaptive immune functions and is a pivotal mediator of inflammatory responses. NF-κB induces the expression of various pro-inflammatory genes, including those encoding cytokines and chemokines, and participates in inflammasome regulation. Moreover, NF-κB plays a critical role in regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells. Deregulated NF-κB activation contributes to the pathogenic processes of various inflammatory diseases

[0091] In some embodiments, the intracellular protein is a Tau protein, which is a member of the Microtubule-Associated Proteins (MAPs). Tau protein is predominantly expressed in the neurons in the brain but is expressed at low levels in oligodendrocytes. Tau proteins’ basic function is believed to be stabilization of microtubule structure in cells. In Alzheimer’s Disease (AD) tau proteins become hyperphosphorylated. Increased phosphorylation causes Tau protein to become insoluble and to self-assemble into paired helical filaments, which form the neurofibrillary tangles of patients with AD. These neurofibrillary tangles destabilize neurons and lead to neurodegeneration, increasing severity of cognitive impairment. Expression of tau protein is increased in damaged cells, such as after heat shock.

[0092] In certain embodiments, the intracellular protein is ERK, Green Fluorescent Protein (GFP), and / or a GFP fusion protein. Erk is a protein that falls under a family of proteins known as MAPK. MAPK families play a critical role in complex cellular programs such as proliferation,P-635623-PC differentiation, development, transformation, and apoptosis. At least three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), Jun kinase (JNK / SAPK) and p38 MAPK. MAPK signaling pathways has been associated in the development of numerous human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers.

[0093] In an embodiment, the intracellular protein is an amyloid beta (Aβ) protein, which is the primary component of neuritic plaques in Alzheimer’s disease (AD), and accumulation of Aβ protein has been considered as the molecular driver of AD pathogenesis and progression. The targeting of tau protein for degradation by administration of a recombinant fusion protein according to an embodiment of the present invention is one form of an anti-Aβ therapy to decrease the formation of neurofibrillary tangles, thereby inhibiting the progression of AD.

[0094] In some embodiments, the intracellular protein is one of multidrug resistance proteins (MRP) family; overexpression of adenosine triphosphate (ATP)-binding cassette ABC) transporters is one mechanism that causes multidrug resistance (MDR) to different classes of anticancer drugs by cancer cells. The MRP family MRP family comprises 13 members, of which MRP1 to MRP9 are the major transporters shown to cause MRD in tumor cells by extruding anticancer drugs out of the cells.

[0095] In an embodiment, the intracellular protein is a protein of the B-cell lymphoma-2 (BCL2) family; Bcl-2 is an anti-apoptotic molecule, whose overexpression has been found in many cancers.

[0096] In some embodiments, the intracellular protein is Cyclin D1 protein, is a nuclear protein that regulates cell cycle progression and whose overexpression has been linked to the development and progression of cancer.

[0097] In an embodiment, the intracellular protein is an AKT protein (also known as Protein kinase B (“PKB”)) comprising three serine / threonine-specific protein kinases, AKT1, Akt12 and AKT3. Overexpression of AKT isoforms in human tumors. Akt1 (E17K), a cancer-derived mutant, is more readily ubiquitinated and phosphorylated than the wild type Akt1.

[0098] In some embodiments, the intracellular protein is a mouse double minute 2 homolog (Mdm2) protein (also known as E3 ubiquitin-protein ligase Mdm2), which is negative regulator of the p53 tumor suppressor, i.e., an inhibitor of p53 transcriptional activation, in addition to being an E3 ubiquitin ligase that binds and ubiquitinates p53, facilitating it for degradation.

[0099] In an embodiment, the intracellular protein is a surviving protein (also called “survivalP-635623-PC protein”) comprising the Bcl-2, inhibitor of apoptosis (IAP), and heat shock protein (HSP) families, whose abnormal expression is associated with a range of biological activities that promote cancer cell survival, proliferation, and resistance to therapy.

[0100] In some embodiments, the intracellular protein is a E2F family protein, i.e., E2F transcription factors that transactivate cell-cycle regulators and pro-apoptotic genes; the E2F family comprises nine known members, including E2F1–E2F3a which are strong transcriptional activators, E2F3b–E2F5 which are passive repressors, and E2Fs 6–8 which have been established as active repressors of transcription.

[0101] In an embodiment, the intracellular protein is hypoxia inducible factor 1 subunit alpha protein (HIF-1alpha), a subunit of the HIF-1 complex, the second subunits of which is HIF-1beta. HIF-1alpha is not present in normal cells but is induced under hypoxic conditions and accumulates after exposure to low oxygen tensions and allows formation of the HIF-1 complex.

[0102] In some embodiments, the intracellular protein is a STAT family protein, which comprises seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6. STAT proteins mainly function as transcriptional regulators, but STAT proteins have also bene shown to be constitutively phosphorylated in many cancers and proliferative diseases. STAT3 has been shown to induce genes, such as genes encoding cyclin D1 and telomerase, that allow sustained proliferation in several human cancers. Phosphorylation of a critical serine residue in the C- terminal domain of STAT proteins has a key role in oncogenic STAT signaling, e.g., serine phosphorylation of STAT5 has been shown to be required for BCR-ABL-induced leukemogenesis and S727phosphorylation on STAT3 was shown to increase the induction of genes involve in the cellular growth and survival that contributed to progression of chronic lymphocytic leukemia.

[0103] In an embodiment, the intracellular protein is tumor necrosis factor alpha (TNF-α), also called tumor necrosis factor, is a factor / cytokine that causes the necrosis of tumors and has been identified as having additional critical functions as a pathological component of autoimmune diseases. Overactivation of TNF-α signaling is associated with chronic inflammation and can cause the development of pathological complications, such as autoimmune diseases.

[0104] In some embodiments, the intracellular protein is an interleukin (IL) family of immunomodulatory proteins (IL-1 to IL-38) that are cytokines with complex immunomodulatory functions, such as cell proliferation, maturation, migration and adhesion and immune cell differentiation and activation. In mammals, IL-1 is a superfamily of eleven structurally similar proteins, all involved in inflammation or its control.P-635623-PC

[0105] In an embodiment, the intracellular protein is natriuretic peptide B (NPPB), a peptide encoded by natriuretic peptide gene (NPPB), a member of the natriuretic peptide family and encodes a secreted protein which functions as a cardiac hormone. NPPB’s biological actions include natriuresis, diuresis, vasorelaxation, inhibition of renin and aldosterone secretion, and a key role in cardiovascular homeostasis. A high concentration of this protein in the bloodstream is indicative of heart failure.

[0106] In some embodiments, the intracellular protein is natriuretic peptide A (NPPA), a peptide encoded by natriuretic peptide gene NPAA. Natriuretic peptides are implicated in the control of extracellular fluid volume and electrolyte homeostasis. NPPA is expressed primarily in the heart, where the expression level is higher in atria than ventricles. Mutations in the NPAA gene have been associated with atrial fibrillation familial type 6.

[0107] In an embodiment, the intracellular protein is C-reactive protein (CRP), which is made by the liver and whose level increases when infection or inflammation is present in the body, e.g., in a chronic inflammatory disease such as rheumatoid arthritis or lupus. A high level CPR has been linked to an increased risk of heart attacks.

[0108] In some embodiments, the intracellular protein is a Transforming growth factor beta 1 (TGFbeta), a member of the Transforming growth factor beta superfamily of cytokines; TGFbeta regulates the growth, proliferation and differentiation of various cell types and is involved in various processes, such as normal development, immune function, microglia function and responses to neurodegeneration, as well as apoptosis.

[0109] In an embodiment, the intracellular protein is alpha-synuclein is a presynaptic neuronal protein that is linked genetically and neuropathologically to Parkinson's disease (PD); insoluble forms of alpha-synuclein accumulate as inclusions in Lewy bodies; alpha-synuclein aggregates are also found in dementia with Lewy bodies (DLB).

[0110] In some embodiments, the intracellular protein is a mucin; mucins are glycoproteins that function inter alia as a protecting lining of the mucosa surface and perform molecular recognition via their glycan chains. Mucin and mucin-like domains have been found to be involved in modulating immune response, inflammation, adhesion, and tumorigenesis. Overexpression of the mucin proteins, mainly MUC1, is associated with several types of cancer.

[0111] In an embodiment, the intracellular protein is p73 protein, which is a homologue of p53, p73 may be a tumor suppressor protein. p73 is activated after DNA damage in a way that is distinct from that of p53.P-635623-PC

[0112] In one embodiment, there are linkers between each component of the fusion protein disclosed herein.

[0113] In one embodiment, the E3 ligase domain is operably linked to a N-terminus of the small protein binding scaffold. In another embodiment, the E3 ligase domain is operably linked to a C- terminus of the small protein binding scaffold. In one embodiment, the anionic polypeptide is operably linked to a C-terminus of the small protein binding scaffold. In another embodiment, the anionic polypeptide is operably linked to a N-terminus of the small protein binding scaffold.

[0114] In another embodiment, there is a reporter or tag at the C-terminus of the fusion protein to facilitate detection of the fusion protein. Various suitable reporters or tags are known in the art.

[0115] In another embodiment, the present disclosure provides a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein. In one embodiment, the cationic agent is a nano-carrier or an ionizable carrier, wherein the nano-carrier or ionizable carrier comprises ionizable-lipid, cationic lipid, polymer, or combination thereof. In one embodiment, the nano-carrier is an ionizable lipid-like nanoparticle (LNP). In one embodiment, the LNP comprises about 30-50% ionizable or cationic lipid, about 30-50% cholesterol, about 10-15% neutral lipid, and about 1-5% PEG-lipid, wherein the percentages are molar percentages.

[0116] In another embodiment, the present disclosure provides a method of targeted intracellular degradation of a target protein, comprising contacting a cell with a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein, wherein the small protein binding scaffold of the fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of the fusion protein.

[0117] In another embodiment, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a composition comprising the fusion protein disclosed herein and a cationic agent that facilitates cytoplasmic delivery of the fusion protein, wherein the small protein binding scaffold of the fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of the fusion protein. In one embodiment, the disease or condition is cancer, sepsis, inflammatory disease, or neurodegenerative disorder. In one embodiment, the cancer is a Ras-driven cancer. In one embodiment, the cancer is pancreatic cancer, non-small cell lung cancer, or colorectal cancer. In one embodiment, the inflammatoryP-635623-PC disease is arthritis. In one embodiment, the neurodegenerative disorder comprises neurofibril degradation.

[0118] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. EXAMPLE 1 Lipid-Mediated Intracellular Delivery of Recombinant bioPROTACs RESULTS Identifying Active N-terminal E3 Domains For bioPROTAC Construction

[0119] A modular bioPROTAC format was established by first screening E3 ligases for degradation activity when fused to the N-terminus of a target-specific DARPin. In total, five E3 domains reported to have high activity were chosen: CHIP, SKP2, SOCS2, SPOP, IpaH9.8 (Figure 2A). For all E3 proteins tested, only their degradation domains capable of recruiting E3 complex proteins or E2 conjugating enzymes were used, and the native substrate binding portions were not included in the bioPROTAC designs. Of note, the IpaH9.8 domain used in this study is not a true mammalian E3 ligase. Instead, the protein is a novel E3 ligase (NEL) derived from Shigella flexneri virulence factors, and its native role is to degrade NEMO and suppress the NF- κB inflammatory response. Nonetheless, IpaH9.8 was included for its outstanding ability to recruit host E2 enzymes for efficient polyubiquitination. With the exception of SKP2, all E3 ligases tested have their natural substrate recognition domain at the N-terminus, and previous groups simply replaced this sequence with desired scaffolds. In contrast to this, the present disclosure chose to install E3 domains at the N-terminus regardless of their natural orientation, as it was previously found that C-terminal placement of polyaspartic acid ApP tags (D25, D30) preserved both expression yields and binding affinity of DARPin scaffolds. Moreover, fusion protein designs where the ApP was placed between the binder and E3 were not considered in this screen. The GFP- binding DARPin 3G124 was used as the targeting domain to allow for a facile fluorescent read- out of successful degradation, and the candidate E3-DARPin formatted bioPROTACs were cloned into pcDNA vectors for characterization.P-635623-PC

[0120] To benchmark bioPROTAC performance, 293T cells were co-transfected with pcDNA plasmids encoding GFP-KRAS and one of the five bioPROTACs at various bioPROTAC:target ratios (Figure 2B). An additional plasmid encoding only the anti-GFP DARPin was also included as a negative control. After 48 hours, cells were analyzed by flow cytometry to assess GFP degradation levels (Figures 2C-2H). No reduction in GFP was observed in cells co-transfected with 3G124 and GFP-KRAS indicating binding alone did not result in degradation. On the contrary, we saw an increase in GFP levels when 3G124 was transfected suggesting DARPin- mediated stabilization of GFP-KRAS. Dose-dependent degradation was observed with SKP2- and SOC2-based bioPROTACs with SOCS2 exhibiting higher activity. Notably, CHIP failed to induce target elimination at any dose, despite its usage in multiple reported bioPROTAC designs. Finally, both SPOP-3G124 and IpaH9.8-3G124 exhibited the highest activity with sharp GFP reductions even at low bioPROTAC amounts. When transfected with the 500ng of GFP-KRAS, just 62.5ng of SPOP and IpaH9.8 bioPROTAC plasmids resulted in 70% and >90% degradation respectively. GFP-KRAS depletion was further validated by Western blotting. Consistent with flow cytometry results, SPOP degraded GFP-KRAS efficiently, while SKP2 and SOCS2 displayed weaker activity (Figure 2I). Interestingly, potent IpaH9.8-induced degradation was observed at low bioPROTAC:target ratios but not at high bioPROTAC levels (Figure 2J) indicating the existence of a hook effect. This phenomenon occurs when PROTAC treatment preferentially drives the formation of POI:PROTAC and PROTAC:E3 binary complexes rather than ternary complexes needed for degradation resulting in a paradoxical reduction in degradation efficiency at higher PROTAC concentrations. As IpaH9.8 binds directly to E2 conjugating enzymes, the hook effect observed here would occur through the formation of E2:bioPROTAC binary complexes rather than through E3 saturation. Based on this screen, both SPOP and IpaH9.8 were chosen as lead E3s, and their developability was established by successful purification as DARPin-fusion proteins that retained substrate binding capabilities (Figure 10). bioPROTAC Delivery with Commercially-Available Transfection Reagents

[0121] Next, 3G124 was replaced with DARPinK2754(K27) to redirect degraders towards undruggable Ras proteins (Figures 3A, 3B). As with 3G124 bioPROTACs, transfection of 293T cells with K27-based bioPROTACs produced potent degradation of co-transfected GFP-KRAS. Here, target destruction is achieved by binding to the KRAS handle. To rule out non-specific degradation, bioPROTACs containing a null K27 mutant with abrogated binding (K27n3) wereP-635623-PC also tested and were not observed to reduce GFP levels (Figures 3C, 3D). After verifying bioPROTAC functionality by transient DNA transfection, whether bioPROTACs would display similar activity as exogenously-delivered purified proteins was investigated. The complete bioPROTAC format includes a target-specific scaffold fused with an N-terminal E3 domain (SPOP or IpaH9.8) and a C-terminal D25 ApP tag. A panel of control proteins was purified to dissect the necessary components of a cytosolically-delivered bioPROTAC system (Figure 3E, Figure 11B). Specifically, a binder-only control (no E3 domain), binding-deficient controls (K27n3), and non- charged (no ApP) variants were cloned and purified. For all proteins, a GFP s11 peptide was included as a reporter of intracellular delivery. The small, 16 amino acid tag does not interfere with protein function and can be used for stringent detection of cytosolic delivery when transfected into cells stably expressing the complementary GFP(1-10) fragment.

[0122] Either SPOP-K27-D25-s11 or IpaH9.8-K27-D25-s11 was complexed with off-the-shelf cationic Lipofectamine 2000 reagent and delivered into 293T cells stably expressing GFP-KRAS (Figure 3F). In agreement with DNA transfection experiments, successful degradation (leftward shift in flow histograms) was observed following intracellular delivery of IpaH9.8-fused bioPROTACs (Figure 3G). However, GFP depletion following treatment with Lipofectamine- complexed SPOP-K27-D25-s11 was not detected. It is unclear why the purified SPOP-based bioPROTAC was unable to deplete GFP-KRAS, as they were able to bind to targets (Figures 10C, 10D). Moreover, chromatograms of SPOP bioPROTACs indicated oligomerization of the E3 ligase (Figures 10C, 11C-11E), suggesting proper protein folding. Thus, it was concluded that the delivery efficiency and / or potency of SPOP-K27-D25-s11 is low and IpaH9.8-based degraders were used for further development.

[0123] Next, the roles of IpaH9.8 and ApP domains on bioPROTAC affinity, intracellular delivery, and target degradation were evaluated. First, binding of the complete bioPROTAC (IpaH9.8-K27-D25-s11) against KRAS was assayed, and the affinity was found to be comparable to that of K27-s11 against KRAS (Figure 12). Therefore, it was concluded that placement of the DARPin between E3 and ApP did not interfere with substrate recognition. To determine if IpaH9.8 and the D25 ApP domains were necessary for degrader functionality, complete Ras-targeting bioPROTACs and a series of controls were either complexed with Lipofectamine 2000 prior to delivery or directly added to 293T GFP-KRAS culture media (Figure 4A). For each condition, 500nM of protein was used, and degradation was quantified by flow cytometry 8 hours afterP-635623-PC treatment. Cytosolic delivery of Lipofectamine-complexed IpaH9.8-K27-D25-s11 resulted in a 46% reduction of GFP-KRAS levels, whereas control proteins lacking E3, binding ability, or ApP could not deplete GFP-KRAS levels. Similarly, the complete bioPROTAC on its own produced no degradation, highlighting the membrane impermeability of these molecules without the aid of a delivery agent.

[0124] To assay cytosolic delivery efficiency of bioPROTACs, 293T GFP(1-10) reporter cells were treated with the same panel of proteins with or without Lipofectamine (Figures 4B, 4C). As expected, only proteins fused with D25 ApPs could complex with Lipofectamine and reach the cytosol. Successful cytosolic delivery was confirmed by increases both in GFP-positivity, a measure of cell transfection efficiency, and in geometric mean fluorescence intensity (MFI), a metric for the amount of protein delivered to cells. The non-binding IpaH9.8-K27n3-D25-s11 control could also be delivered into cells with the same efficiency as active bioPROTAC but was unable to degrade targets. It was confirmed that this was due to the protein’s inability to polyubiquitinate KRAS (Figure 13). Hence, it was shown that the E3 ligase, target-specific binder, and negatively-charged ApP are fundamental requirements for protein degradation in this bioPROTAC platform.

[0125] Since Lipofectamine is a readily-available transfection reagent, Lipofectamine-mediated bioPROTAC transfection was further characterized for utility as a research tool. Following incubation with Lipofectamine:bioPROTAC, a dose-dependent decrease in GFP levels was observed in 293T GFP-KRAS, and at 8 hours post-treatment, a maximum degradation efficiency of 45% was reached (Figure 4D). This coincided with a peak delivery efficiency of ~22% and a 1.85-fold increase in MFI compared to untreated cells (Figures 4E, 4F). Both degradation and delivery efficiencies were maximized at 125nM bioPROTAC complexed with 2µL of Lipofectamine. Furthermore, both degradation efficiency and delivery efficiency were found to be time dependent (Figures 14A-14C). Lipofectamine-delivered bioPROTACs were also compared to commonly-used RNAi methods, and 293T GFP-KRAS cells were transfected with two different KRAS-targeting siRNA using Lipofectamine RNAiMAX (Figure 14D). In this head-to-head comparison, bioPROTACs displayed greater than 3 times faster degradation kinetics than siRNA (Figure 14E).

[0126] Taken together, purified ApP-tagged bioPROTACs could serve as a powerful research tool when paired with easily-accessible cationic transfection reagents and would facilitate biological interrogation at much shorter time scales without the need to genetically modify cells. It was alsoP-635623-PC shown that this knockdown approach circumvents one of the intrinsic limitations of siRNA (slow kinetics) for potential therapeutic applications. As another advantage over RNA, the stability of purified bioPROTACs was tested under simple storage conditions and it was verified that they displayed no loss in activity after four weeks when stored in PBS at 4°C (Figure 15). Identifying an Optimal LNP Formulation For Intracellular Delivery of bioPROTAC Protein

[0127] It is important to note that despite improved target knockdown compared to siRNA, complete degradation was not achieved by Lipofectamine-mediated transfection of bioPROTACs. It was hypothesized that this incomplete elimination by Ras-targeting degraders was due to low Lipofectamine transfection efficiency. To improve intracellular protein delivery, LNPs was explored for bioPROTAC encapsulation, as they exhibit high drug loading capacity and promote endosomal escape for enhanced cytosolic access. Initially, to develop LNPs for bioPROTAC encapsulation, three base formulations were used (Table 1). Proteins were formulated into LNPs by microfluidic mixing of a bioPROTAC-containing aqueous phase and lipid- / excipient- containing ethanol phase (Figure 5A). All three formulations performed better than Lipofectamine 2000, validating initial reasoning for using LNPs. Excitingly, treatment with the K1 formulation led to 95% GFP-KRAS elimination at a 200nM bioPROTAC dose (Figures 5B, 5C). Interestingly, while the B6 formulation was previously optimized for DARPin-ApP delivery, it only displayed a degradation efficiency of 60%. To study how LNP composition contributed to LNP:bioPROTAC formation and subsequent degradation efficiency, a library of LNP formulations was generated based off of the K1 formulation which includes C12-200 as the ionizable lipid (Table 2). Formulations incorporating 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the helper lipid exhibited the highest degradation efficiency (Figure 16A). Ultimately, the K1 formulation was chosen as the lead LNP due to its functional potency and physical properties. The formulation retained high degradation efficiency up to one week following formulation (Figure 16B), had a relatively small hydrodynamic diameter of 167nm, and exhibited near-complete encapsulation of protein cargo (Figures 16C-16E, Table 3). TABLE 1. Initial base LNP formulations for bioPROTAC formulation and delivery. Identifier K1 C1 B6P-635623-PC Helper lipid (mol %) 19.0 10.0 10.0TABLE 2. Library of LNP formulations screened for degradation efficiency. C14PEG- C14PEG- C12-200 Total 2000 C12-200 DOTAP Total helper lipid helper lipid cholesterol 2000 (mol DOTAP (mol helper lipid cholesterol (mol (mol (mol (mol Identifier type (mol ratio) (mol ratio) (mol ratio) ratio) (mol ratio) ratio) (mol fraction) (mol fraction) fraction) fraction) fraction) fraction) 0 0 0 0 0 0 0 0 0TABLE 3. Physical characterization of select bioPROTAC LNP formulations. LNP Cargo Size, average diameter PDI Zeta potential (mV) (nm)

[0128] The degradation dose response of K1:bioPROTAC was investigated in 293T GFP-KRAS cells by flow cytometry, and a half-maximal degradation (DC50) of 19.5nM was calculated at 8- hours post-treatment (Figure 5D). The acute cytotoxicity of LNP:bioPROTAC in 293T cells was determined using a lactate dehydrogenase (LDH) assay, and cell viability was found to be >90% at protein concentrations up to 100nM (Figure 5E). The increased target clearance by LNP:bioPROTAC was accompanied by a dramatic improvement in delivery efficiency. AfterP-635623-PC treatment with 200nM LNP-delivered bioPROTAC, 49% of 293T GFP(1-10) were GFP-positive, representing a two-fold increase in cytosolic delivery efficiency over Lipofectamine (Figure 5F). The amount of protein delivered was commensurate, with LNP:bioPROTAC achieving a 3-fold change in MFI over the control group (Figure 5G). For both LNP and Lipofectamine delivery approaches, it was noticed that the degradation efficiency was greater than the delivery efficiency. This can be attributed to the non-stoichiometric mechanism of bioPROTACs in which one degrader molecule can catalyze the destruction of many target molecules. Moreover, the limit of detection for split GFP assays is in the low nanomolar range, meaning split GFP may lack sufficient sensitivity to reflect highly-active degradation activity.

[0129] Next, it was determined whether K1:bioPROTAC could degrade endogenous Ras as opposed to GFP-KRAS fusion proteins used in characterization studies. To this end, wild-type (WT) 293T cells were treated with LNPs encapsulating either K27-D25-s11, IpaH9.8-K27-D25- s11, or IpaH9.8-K27n3-D25-s11. Lysates from treated cells were analyzed by Western blotting and probed with a pan-Ras primary antibody (Figure 5H). Closely mirroring flow cytometry results, incubation with 100nM K1:bioPROTAC for 8 hours resulted in up to 80% endogenous Ras degradation as calculated by band densitometry (Figure 5I). In accordance with the proposed mechanism of action, neither B6:K27-D25-s11 nor K1:bioPROTAC(null) led to reductions in Ras levels owing to a lack of E3 activity and binding affinity respectively, and the bioPROTAC protein alone did not induce target degradation due its inability to enter cells without a delivery vehicle. A panel of cancer cells was also treated with LNP:bioPROTAC to validate the generalizability of the protein degrader disclosed herein in multiple cell types. A similar pattern of degradation was observed in HCT116 colorectal cancer cells, A549 lung cancer cells, and HT1080 fibrosarcoma cells. On average, the Ras degradation efficiency in these cell lines ranged from approximately 40% to 55% (Figure 17). Target Degradation Kinetics With LNP-Delivered bioPROTAC

[0130] To study the degradation kinetics of LNP-delivered bioPROTACs, 293T GFP-KRAS cells were further engineered to express iRFP-CaaX, a fluorescent membrane marker. These dual- reporter cells enabled simultaneous degradation analysis and live-cell tracking via an orthogonal mask. As a direct comparison to direct protein delivery, a treatment group receiving LNP- encapsulated mRNA encoding IpaH9.8-K27 (bioPROTACmRNA) was added. Cells were incubated with LNPs at dosages producing maximum degradation (Figures 5D, 18), and GFP was monitored for 12 hours. As expected, cell-only controls exhibited no changes in GFP fluorescence intensityP-635623-PC (Figures 6A, 6D). Meanwhile, both LNP-mediated bioPROTAC protein delivery (Figures 6B, 6E) and bioPROTAC mRNA delivery (Figures 6C, 6F) resulted in rapid and robust GFP clearance, reaching maximum degradation efficiency in 5 hours. The target half-life following protein delivery was calculated to be 97 minutes, while the half-life for mRNA was calculated to be 146 minutes. Treatment with either null bioPROTAC or binder only controls did not lead to degradation and actually resulted in an apparent stabilization of the target protein (Figure 19).

[0131] For many therapeutic applications, prolonged degradation is desirable, so it is critical to understand the duration of bioPROTAC activity following LNP-mediated delivery. Flow cytometry was used to analyze LNP-treated cells at various time points up to 48 hours post- delivery with either bioPROTACproteinor bioPROTACmRNA(Figure 6G). At both low and high doses, proteins produced sharp GFP depletion within 4 hours. The maximum degradation efficiency was nearly identical for both modalities, reaching up to ~95% target reduction by 8 hours. Recovery of GFP-KRAS levels was similarly comparable between protein and RNA. Under treatment washout conditions (LNP-containing media replaced with fresh media at 4 hours), complete recovery was observed by 48 hours. Conversely, sustained presence of LNPs in solution (no washout) resulted in only 30-40% recovery at 48 hours. Global Proteome Response to bioPROTAC Treatment

[0132] To understand how bioPROTACs were affecting protein levels globally, shotgun proteomics was performed following degrader delivery. Cells were either left untreated, treated with K1:Ras bioPROTAC protein, or transfected with LNP:bioPROTAC mRNA. A group receiving K1:null bioPROTAC protein was also included to examine the specificity of the present degraders. Cells were treated for 8 hours, and extracted proteins were subjected to tandem mass spectrometry (MS). Label-free quantification resulted in 4752 uniquely-identified proteins common to all treatment groups. After filtering proteins with PSM > 24, a final dataset of 3827 proteins was used for downstream analysis. Proteins were considered differentially-expressed if the log2 fold-change (log2fc) between treated and untreated groups was either ≤ -1 or ≥ 1 and the p-value < 0.05. In both mRNA bioPROTAC and protein bioPROTAC groups, NRAS and KRAS abundance was lower compared to no treatment, although KRAS did not meet the log2fc threshold in the protein-treated group (Figures 7A, 7B). Knockdown of NRAS and KRAS was not observed in cells treated with null bioPROTAC (Figure 7C). These results matched the expected depletion pattern in samples treated simultaneously and analyzed by Western blotting (Figure 20A). In addition to NRAS, several proteins were also identified significantly downregulated in activeP-635623-PC bioPROTAC treatment groups but in the null bioPROTAC treatment group (Figure 7D). Amongst these, members of the trimeric G-protein family: GNAI1, GNAI2, and GNAI3 were found to be depleted by both bioPROTAC protein and mRNA treatment. These proteins are structurally homologous to Ras-family proteins, containing P-loop and G-Box motifs also found near the DARPinK27-KRAS binding site. Thus, it is suspected that guanine nucleotide-binding protein G(i) subunit alpha is a bona-fide off-target for DARPinK27. The relationship between the remaining proteins is less clear. However, all of them are membrane-associated. Furthermore, CHMP4A, MYO1B, and MYO1D are implicated in endosomal trafficking processes. Specifically, CHMP4A is known to facilitate lysosomal degradation of ubiquitinated membrane proteins, and ubiquitination-dependent lysosomal trafficking has been documented for other surface receptors. These data raise the possibility that other degradation pathways including endosomal-lysosomal sorting contribute to bioPROTAC-mediated target depletion, at least in the case for membrane- associated targets.

[0133] It was also noticed that a high percentage of shared down proteins between mRNA and null bioPROTAC were not identified in the protein bioPROTAC group (Figure 7D). To better understand these downregulated proteins, Gene Ontology (GO) biological process (BP) enrichment analysis was performed and found that many members of this group mapped to RNA splicing machinery (Figure 20B). Prior reports have found that IpaH9.8 binds to and inhibits U2AF splicing factors. In addition, 18 other U2AF-interacting proteins were downregulated, suggesting co-regulation by the IpaH9.8 domain (Figure 20C). Although the present bioPROTAC does not include the IpaH9.8 substrate-recognition domain, U2AF binding was found— counterintuitively—to occur through the IpaH9.8 C-terminal domain. Interestingly, the active bioPROTAC protein induced non-significant decreases in U2AF despite also having the IpaH9.8 catalytic module. This mitigated off-target effect could be because the protein’s targeting domain redirects its activity away from U2AF. In addition, its intracellular concentration at 8 hours could be lower compared to mRNA-expressed bioPROTAC.

[0134] Despite effectively degrading NRAS and KRAS, mRNA treatment led to substantially more differentially-regulated proteins compared to bioPROTAC protein delivery. Some of these proteins were assigned to mRNA processing pathways via GO enrichment analysis (Figure 7E) and are likely cellular responses to exogenous mRNA delivery. Molecular function enrichment analysis was performed on significant mRNA treatment knockdowns, and excluding Ras, an additional 12 GTPases that were degraded by mRNA bioPROTAC was uncovered. This findingP-635623-PC was further investigated and the fold-changes of all GTPases found to be downregulated either in mRNA or protein bioPROTAC treatment groups were compared. With the exception of RhoB, both protein and mRNA-mediated degradation of Ras and Ras-like proteins were correlated (Figure 7F). Interestingly, a subset of GTPases with little homology to Ras-family proteins was identified, including α- and β-tubulins, that were strongly downregulated by mRNA bioPROTAC but not by recombinant protein bioPROTACs, indicating plausible modality-specific degradation off-targets. Moreover, transfection with mRNA produced many additional downregulated proteins that were not accounted for by pathway or molecular function enrichment. Taken together, these results demonstrate that protein knockdown profiles can differ greatly depending on whether bioPROTACs are delivered as proteins or expressed from mRNA. Mechanistic Exploration of bioPROTAC Target- and Self-Degradation

[0135] Autoubiquitination and self-degradation are major concerns with our bioPROTAC design. This is because the IpaH9.8 NEL domain used in the present constructs lacks the native auto- inhibitory mechanism present in full-length IpaH9.8 critical to preventing self-targeting. To investigate the fate and degradation mechanism of the present bioPROTACs disclosed herein, a catalytically-dead variant was produced by mutating the catalytic cysteine residue in IpaH9.8 NEL to alanine. IpaH9.8C337A-K27-D25-s11 was purified and its inactivation confirmed via in vitro ubiquitination assays (Figures 21A-21C). It was found that this inactivation was not due to changes in binding affinity (Figure 21D). Furthermore, wild-type bioPROTACs were prone to autoubiquitination (Figure 21E), and this self-targeting was completely abolished in the C337A mutant. Upon delivery into GFP(1-10) reporter cells, the IpaH9.8C337Avariant displayed significantly higher apparent transfection efficiency compared to bioPROTACs fused to IpaH9.8WT(Figures 22A-22C). This increased GFP signal indicates higher levels of bioPROTAC in the cytosol. As there is only a single amino acid difference between the two proteins, this discrepancy should not stem from differences in cargo encapsulation or delivery efficiencies. Instead, it is proposed that the present bioPROTACs self-ubiquitinate upon intracellular delivery, reducing the cytosolic pool of degrader proteins. Addition of MG-132, a peptide proteasome inhibitor to culture media did not significantly increase GFP complementation levels but did rescue degradation of both GFP-KRAS and endogenous Ras (Figures 22D, 22E). Taken together, these data indicate that bioPROTAC-mediated target degradation does occur via the ubiquitin- proteasome pathway. On the other hand, bioPROTAC self-degradation depends on E3 catalyticP-635623-PC activity but not proteasomal degradation despite autoubiquitination. This points to another mechanism of bioPROTAC self-destruction such as ubiquitin-mediated autophagy or through the endosome-lysosome pathway. Modularity of the bioPROTAC Format

[0136] To examine the ability of the bioPROTAC platform disclosed herein to degrade diverse targets, K27 was replaced with alternate DARPin sequences, redirecting their activity towards other endogenous substrates (Figure 8A). In one embodiment, DARPins that bind extracellular signal-regulated kinase 1 / 2 (Erk 1 / 2), c-Jun N-terminal kinases (Jnk), or B-cell lymphoma-extra large (Bcl-xL) were chosen, as these proteins are promising therapeutic candidates being pursued for chemical inhibition. All four alternate bioPROTACs could be purified with good yield (Figure 8B). The bioPROTACs were encapsulated in LNPs using the K1 formulation and delivered to either 293T or A549 cells. Clear depletion of Jnk and Erk bands were observed by western blotting following treatment with J1 / 2_2_25 and EpE89 respectively (Figure 8C, lanes 3 and 4). By contrast J1 / 2_2_3 and 012_F12 failed to noticeably degrade their respective targets: Jnk and Bcl- xL (Figure 8C, lanes 2 and 5). Notably, while both J1 / 2_2_25 and J1 / 2_2_3 are N2C-formatted DARPins identified from separate screens against Jnk, they produced different degradation outcomes. When degradation was observed, it was highly specific, as each unique bioPROTAC degraded only their intended targets while sparing the other surveyed proteins. This success rate for alternate bioPROTACs was achieved with no additional engineering of the bioPROTAC scaffold, and alternate degraders were easily produced by “plug-and-play” cloning. Thus, in agreement with previous reports of cell-expressed bioPROTACs, it is concluded that the purified, ApP-tagged degrader format disclosed herein is modular and exhibits a high degree of design flexibility.

[0137] It was next examined whether the bioPROTAC format disclosed herein could degrade targets localized to various intracellular compartments. Both Ras- and GFP-targeting degraders already demonstrated potent activity against GFP-KRAS and endogenous Ras, both of which associate with the inner leaflet of the plasma membrane. In addition, the present GFP-targeting bioPROTAC (IpaH9.8-3G124-D25-s11) was tested against a panel of cell lines stably expressing GFP-fusions. Using the K1 formulation for cytosolic delivery, bioPROTACs effectively eliminated GFP localized to the nucleus (GFP-MIS12, GFP-TRF1), cytosol (GFP-SPC25), and mitochondria (GFP-MITO). Representative fluorescent images showed near-complete degradationP-635623-PC just 8 hours after LNP incubation (Figure 8D). This result was further confirmed by flow cytometry analysis. Again, GFP-targeting bioPROTACs completely silenced fluorescence signal, whereas Ras-targeting bioPROTACs, used as a negative control, had no effect on target levels (Figure 8E). Inhibition of Pancreatic Tumor Cells with a Ras-Degrading bioPROTAC

[0138] Finally, to demonstrate a potential therapeutic application of LNP: bioPROTACs, the antiproliferative effects of Ras-degrading bioPROTACs when delivered into MIA PaCa-2, a PDAC line harboring a KRAS G12C driver mutation was examined. First, it was confirmed that Ras-targeting bioPROTACs were functional once delivered into MIA PaCa-2. Cells were incubated with K1 LNP formulations of active and null bioPROTAC proteins or bioPROTAC- encoding mRNA, and lysates were analyzed by Western blotting (Figure 23A-23B). Following LNP treatment, Ras was depleted in a dose-dependent manner with IpaH9.8-K27 mRNA or IpaH9.8-K27-D25-s11 protein, but not with IpaH9.8-K27n3-D25-s11 protein (Figures 9A-9C). Phosphorylated Erk (pErk), a key effector in the canonical MAPK signaling pathway was also probed and it was observed that it was depleted in tandem with Ras degradation. Next, cells were treated with K1:bioPROTAC, and proliferation was monitored using an impedance-based confluency assay. As expected, neither IpaH9.8-K27-D25-s11 nor IpaH9.8-K27n3-D25-s11 protein alone (no LNP) affected MIA PaCa-2 proliferation (Figure 9D). By contrast, at 24 hours post-treatment, K1:IpaH9.8-K27-D25-s11 led to 46% and 79% reduced growth at low (56nM) and high (140nM) bioPROTAC concentrations respectively. These effects were statistically significant when compared to treatment with K1:IpaH9.8-K27n3-D25-s11, indicating that anti-proliferative effects were due to Ras targeting. These results were compared against bioPROTAC mRNA treatment, which showed comparable viability inhibition at 150ng / mL and 300ng / mL mRNA. These results are consistent with observed Ras degradation at the two highest mRNA doses.

[0139] Despite promising growth inhibition with K1:bioPROTAC, it was noticed some viability loss in MIA PaCa-2 cells treated with K1-formulated null bioPROTAC, indicating non-specific toxicity. To alleviate toxicity arising from the particles themselves, variations of the K1 formulations substituting the C12-200 lipid with a series of commercially-available and previously-published ionizable lipids were screened (Figure 24). It was found that LNPs incorporating SM-102 retained anti-proliferative effects, exhibiting ~70% growth inhibition while reducing non-specific toxicity to 18% at a 140nM protein dose (Figure 24E). Thus, Ras degradation by LNP-delivered recombinant bioPROTACs displays therapeutic effects byP-635623-PC degrading oncogenic proteins. DISCUSSION

[0140] In summary, the present disclosure discloses a modular recombinant bioPROTAC platform capable of on-demand, targeted protein degradation using lipid nanocarriers. In one example format, incorporating an N-terminal IpaH9.8 NEL and C-terminal ApP demonstrated robust, low nanomolar activity in 7 different cell lines and could be easily reprogrammed to polyubiquitinate diverse substrates including GFP, Ras, Erk, and Jnk. Strikingly, the present bioPROTACs were active in various subcellular compartments including the cytosol, membrane, mitochondria, and nucleus. Finally, the biologic-based degraders disclosed herein were highly potent, exhibiting target elimination within hours of treatment. From a manufacturing standpoint, this format presents several key advantages. Firstly, the bioPROTACs are easily expressed using inexpensive bacterial cultures, enabling low-cost and accessible prototyping / testing of new degraders. Secondly, protein-based bioPROTACs exhibited no loss in activity for several weeks, are not susceptible to nuclease activity, and are amenable to typical storage conditions.

[0141] For Ras-targeting, it was found that the general trend for degradation rate was, in increasing order: siRNA < DNA < mRNA < protein. While this is in line with the expected result, it is noted that it is difficult to make direct comparisons of degradation rates between these modalities. For example, protein expression from plasmids is heterogeneous, and both DNA and mRNA can generate many protein molecules per nucleic acid obfuscating direct assessments against protein delivery. Moreover, siRNA-mediated degradation is dependent on the stability of their target protein and can have vastly different depletion rates. Nonetheless, rapid degradation was consistently observed using the present LNP-delivered bioPROTAC format and there was a target half-life of ~1.5 hours following treatment. Only small-molecule systems such as DTag, AID, or conventional PROTACs display faster kinetics (<30 minutes), but these are burdened by many challenges as previously mentioned. To further highlight differences between modalities, an apparent hook effect was only observed with pcDNA bioPROTAC transfection. This is likely due to the inclusion of a strong CMV promoter and a 5’ Kozak sequence. These elements drive excessive bioPROTAC production to levels that are likely not reached by other delivery methods.

[0142] In addition to kinetic differences between degradation modalities, varying target specificity was also found depending on how the bioPROTAC was introduced. When transfected with mRNA encoding a Ras-targeting bioPROTAC, many non-target knockdowns were identified via MS / MS global proteome analysis. Comparatively, delivery of the same bioPROTAC as a recombinantP-635623-PC protein induced fewer off-target effects. While some of the additional downregulated targets in the mRNA group were identified as RNA-processing proteins, many others were not reliably explained. Taken together, the present results support the idea that for some bioPROTACs, mRNA could induce more off-target degradation compared to protein delivery. Recently, pseudouridine- substituted mRNA was found to increase protein mistranslation, and it is not clear if mutations in the DARPin variable region could generate off-target degraders from modified mRNA.

[0143] Although it has been successfully exploited as a C-terminal degradation domain, CHIP displayed no activity when fused to the N-terminus. This suggests that some E3 domains are more flexible than others for bioPROTAC development. Interestingly, it was found that IpaH9.8-K27 was better than the previously-reported SPOP-K27 at eliminating Ras (Figures 3C, 3D). A similar result was recently published, showing that IpaH9.8 was more effective than SPOP at degrading GFP. In the present disclosure, this observation was confirmed, noting increased degradation rates for IpaH9.8 (1.5 hours) versus SPOP (>2.5 hours). In addition, it was found that SPOP-K27 could not be converted into an exogenously-delivered degrader (Figure 3G). It is hypothesized that this failure is due to a lower intrinsic potency and / or a reduced ability to complex with lipids caused by SPOP oligomerization. Thus, it is concluded that IpaH9.8 is a highly-active E3 domain which can be purified from bacterial cultures and remains active following lipid-mediated cytosolic delivery. The IpaH9.8 NEL is also tolerant to positioning within chimeric proteins, as it is now confirmed to be active in both N-terminal and C-terminal designs.

[0144] Towards extending bioPROTACs for clinical applications, polymeric nanogels have been harnessed for cytosolic antibody delivery and target degradation via TRIM-away. This method results in 50% degradation between 4-6 hours post-treatment. In another approach, the ZF5.3 CPP was appended to a BCL11A-targeting, SPOP-based degrader to enhance bioPROTAC internalization. With this system, 70% depletion of BCL11A was achieved within 12 hours using a final protein concentration of 10µM. In comparison to these two recent methods, the LNP / bioPROTAC-ApP approach described herein is both faster and more potent, with the ability to reach >90% degradation within 5-6 hours using less than 100nM of protein. This efficiency was made possible through the combination of a highly-active IpaH9.8 module, negatively-charged ApP, and a compatible LNP vehicle.

[0145] From LNP screening results, it is noted that DOPE is a critical factor for bioPROTAC LNP performance, and improved LNP stability and increased degradation in formulations incorporating DOPE as a helper lipid were observed. This is consistent with the DOPE’s known propensity toP-635623-PC enhance endosomal escape by membrane fusion. During therapeutic studies in MIA PaCa-2 PDAC cells, it was noticed that some LNPs caused non-specific cytotoxicity at high doses which were alleviated by changing the ionizable lipid. This finding underscores the importance of tuning LNP properties based on cell line dependent responses. Crucially, future studies in animal models should carefully screen LNP formulations to simultaneously enhance target tissue uptake while reducing non-specific toxicity. Simultaneously, engineering of bioPROTACs for stronger degradation can lower the therapeutic dose, further reducing off-target effects.

[0146] In terms of cargo engineering, DARPins were highly-tolerant as chimeric fusion proteins, highlighting their promise for targeted degradation. In addition, the present disclosure describes degraders from various DARPin formats including N3C (3G124, K27) and N2C (J1 / 2_2_3, J1 / 2_2_25, EpE89) scaffolds.

[0147] Critically, it was found that IpaH9.8-fused bioPROTACs were subject to autoubiquitination leading to self-destruction upon cytosolic delivery. This effect did not restrict LNP:bioPROTAC efficacy in vitro, as potent degradation was still observed in cell assays. However, autoubiquitination could limit the ability of bioPROTAC to accumulate to therapeutic doses in vivo. One potential solution to relieve self-inhibition would be to identify lysine residues on the bioPROTAC (18 total on the Ras degrader) that are prone to self-targeting. These residues can then be selectively mutated to either alanine or arginine, rendering bioPROTACs resistant to self-degradation. Such an approach has demonstrated success in stabilizing other biodegraders.

[0148] In one embodiment, the present disclosure used DARPins as model binders owing to their stability. Additionally, methods for screening diverse DARPins are well-established. Other binding scaffolds including affibodies, nanobodies, and monobodies may also be tested in the format disclosed herein for even broader versatility and application scope. With the ever- expanding collection of small protein scaffolds selected for antibody-like affinity and specificity, it is expected that bioPROTACs will become more attractive as a therapeutic modality. Importantly, biodegraders expand the druggable proteome by removing the requirement for binders to have an inhibitory effect. The ability to deliver these protein-based degraders into cells serves as a promising avenue towards the treatment of many intractable diseases. METHODS Cloning

[0149] All DNA fragments used in this study were synthesized as gBlocks (IDT; Coralville, IA),P-635623-PC and codon optimization was performed using the IDT online codon optimization tool. For mammalian cell transfection experiments, gBlocks were codon-optimized for human cell line expression, and genes were inserted into a CMV-driven pcDNA3.1 vector with a 5’ Kozak sequence. Similarly, the lentivirus transfer plasmid was codon-optimized for human cell line expression. For bacterially-produced proteins, genes were codon-optimized for E. coli K12 expression and inserted into sortase tag-expressed protein ligation expression plasmid (pSTEPL). For cloning into pcDNA3.1 and pSTEPL, plasmids were linearized by double digestion with the appropriate restriction enzymes followed by backbone isolation using a QIAquick gel extraction kit (Qiagen; Germantown, MD). Plasmids for bacterial transformation and mammalian transfection were purified by Qiagen miniprep and Qiagen endotoxin-free maxiprep kits respectively. All plasmids were submitted for Sanger sequencing to confirm successful cloning of the correct protein sequence.

[0150] To generate the E3-deficient binder-only control, the anti-GFP DARPin, 3G124, was cloned into the linearized pcDNA3.1 backbone between KpnI and EcoRI using In-Fusion Snap Assembly master mix (Takara Bio; San Jose, CA). To produce the bioPROTAC mammalian- expression plasmids, an SPOP167–374-3G124 encoding gBlock was first inserted into the pcDNA3.1 backbone between KpnI and EcoRI by In-Fusion cloning. A GS-rich linker was included between SPOP and 3G124. In addition, a BamHI restriction site was designed into the gBlock between the two proteins for convenient cloning of alternate degradation domains. The remaining E3 sequences: SKP22-147, IpaH9.8254-545, SOCS2143-198, and CHIP128-303were inserted into this plasmid between KpnI and BamHI by In-Fusion cloning. To generate Ras-targeting bioPROTAC mammalian expression vectors, gBlocks for SPOP167–374-DARPinK27 and SPOP167–374- DARPinK27n3 were first cloned into pcDNA3.1 between KpnI and EcoRI. Again, both GS-rich linkers and BamHI sequences were included between the E3 domain and DARPin domain, so that IpaH9.8254-545 could be easily substituted for SPOP. The target plasmid: pcDNA3.1 GFP-KRAS was created by cloning KRAS into an eGFP-containing plasmid between BsrGI and EcoRV.

[0151] pSTEPL plasmids for the expression of K27-D25-s11, K27-D30-s11, K27n3-D25-s11, and K27n3-D30-s11 in E. coli were previously produced. The DARPin domains in these plasmids were replaced with 3G124 to produce 3G124-D25-s11 and 3G124-D30-s11. To generate SPOP-3G124- D25-s11, a gBlock encoding SPOP167–374-3G124 was inserted into pSTEPL plasmids between NdeI and XhoI to retain the C-terminal ApP and s11 sequences. Using the resulting modular template, the remaining bioPROTAC expression plasmids were produced. Specifically, theP-635623-PC IpaH9.8254-545NEL sequence (wild-type or C337A mutant) was cloned between NdeI and BamHI, and the other target-specific DARPins (or null control): K27, K27n3, J1 / 2_2_3, J1 / 2_2_25, EpE89, and 012_F12 were cloned between BamHI and XhoI. To remove the D25 ApP sequence, pSTEPL plasmids were first digested with XhoI and AgeI. The backbones were then purified by gel extraction, and finally, a gBlock encoding the GFP s11 sequence was re-inserted at the same site. All proteins contained GS-rich linkers between E3, binding, ApP, and s11 reporter domains.

[0152] The GFP-KRAS lentiviral transfer plasmid was made by first linearizing the pLX304 vector (Addgene #25890) by PCR. Then, a gBlock encoding eGFP-KRAS was cloned into the pLX304 backbone using an In-Fusion HD cloning kit. The gene fragment contained a 5’ Kozak sequence and a GS-rich sequence between eGFP and KRAS proteins. To generate the iRFP-CaaX transfer plasmid, the iRFP sequence was amplified from DEST-H2B-iRFP670 (Addgene #90237) and cloned into a pHR lentiviral backbone upstream of a C-terminal CaaX sequence. Protein Expression and Purification

[0153] For all proteins, bacteria were cultured in 2YT autoinduction media including trace elements (Formedium; Norfolk, United Kingdom).

[0154] The following proteins were expressed in Shuffle T7 Express competent E. coli (C3029J, New England Biolabs; Ipswich, MA): SPOP-3G124-s11, SPOP-3G124-D25-s11, IpaH9.8- 3G124-D25-s11, SPOP-K27-D25-s11, SPOP-K27n3-D25-s11, IpaH9.8-K27-D25-s11, IpaH9.8- K27n3-D25-s11, IpaH9.8-J1 / 2_2_3-D25-s11, IpaH9.8-J1 / 2_2_25-D25-s11, IpaH9.8-EpE89- D25-s11, and IpaH9.8-012_F12-D25-s11. All other binders and bioPROTACs were expressed in T7 Express competent E. coli (C2566, New England Biolabs). Additionally, T7 Express E. coli previously transformed with pSTEPL eGFP was used in this study for recombinant GFP expression. Expression cultures for SPOP-3G124-D25-s11, SPOP-K27-D25-s11, and SPOP- K27n3-D25-s11 were grown at 25°C for 48 hours. Expression cultures for IpaH9.8-J1 / 2_2_3-D25- s11, IpaH9.8-J1 / 2_2_25-D25-s11, IpaH9.8-EpE89-D25-s11, and IpaH9.8-012_F12-D25-s11 were grown at 30°C for 24 hours. All other proteins were expressed at 37°C for 24 hours. Cultures were grown in baffled flasks and shaken at 160-180 rpm. Using the STEPL bioconjugation / purification method, proteins were eluted from HisPur cobalt resin (Thermo Fisher Scientific) with triglycine (GGG). For some assays such as encapsulation or binding studies, dye- or biotin-labeled GGG peptides were used for C-terminal bioconjugation.

[0155] Following elution, proteins were further purified by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva; Marlborough, MA). Fractions were pooledP-635623-PC and concentrated using Amicon Ultra centrifugal filters with a 10 kDa molecular weight cut-off (MilliporeSigma; Burlington, MA). The purity of the final product was characterized by SDS- PAGE, and protein concentration was determined using the BCA assay. Protein stocks were stored at -80°C for later use. Cell Culture

[0156] The following cell lines: 293T, 293T GFP-KRAS, 293T GFP-KRAS / iRFP-CaaX, 293T GFP(1-10), A549, and HT1080 were either obtained from our own stocks or produced for this study. HeLa cells stably expressing HaloTag-GFP-MITO, HaloTag-GFP-SPC25, HaloTag-GFP- MIS12, or HaloTag3x-GFP-TRF1 and U2OS cells stably expressing HaloTag3x-GFP-TRF1 were generously gifted by Michael Lampson. All of the above cell lines were cultured in DMEM (Thermo Fisher Scientific; Waltham, MA) supplemented with 1% pen-strep and 10% FBS. The colorectal cancer cell line, HCT116 was gifted by Michael Farwell and cultured in McCoy’s 5A medium (Thermo Fisher Scientific), and the pancreatic cancer line, MIA PaCa-2 was gifted by Gregory Beatty and cultured in RPMI (Thermo Fisher Scientific). Both McCoy’s 5A and RPMI media were supplemented with 1% pen-strep and 10% FBS. All cells were grown in a humidified incubator maintained at 37°C and 5% CO2 conditions. All native cell lines used in this study were authenticated by short tandem repeat (STR) profile analysis by the Penn Genomic and Sequencing Core. Stable Cell Line Engineering

[0157] Lentivirus for GFP-KRAS transduction was produced using standard techniques. Briefly, equal amounts of pLX304 GFP-KRAS, pMD2.G (Addgene #12259), and psPAX2 (Addgene #12260) were transfected into 293T with Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. The virus-containing media was collected 48-hours later, centrifuged to remove cell debris, and passed through a 0.45µm PES syringe filter. Either 1mL, 0.5mL, or 100µL of clarified lentivirus supernatant was added to separate 293T cells seeded in 6- well plates in complete DMEM. After 24 hours, the culture media was exchanged for fresh DMEM. Once cells were confluent, GFP-positive populations were isolated with the assistance of the CHOP Flow Cytometry Core using a FACSJazz sorter (BD Biosciences; Franklin Lakes, NJ).

[0158] To generate 293T GFP-KRAS / iRFP-CaaX dual reporter cells, 293T GFP-KRAS cells were first plated overnight in a 6-well plate at 300,000 cells / well. The following day, culture media was replaced with complete DMEM containing 8µg / mL Polybrene, and 300µL of iRFP-CaaXP-635623-PC lentivirus was added to cells and allowed to incubate overnight. Cells were grown until confluent and sorted with the assistance of the CHOP Flow Cytometry Core using a MoFlo Astrios cell sorter (Beckman Coulter; Brea, CA) for dual GFP / RFP positivity. mRNA Synthesis

[0159] Production of mRNA was carried out by the Penn Institute for RNA Innovation mRNA Core. The coding sequence of IpaH9.8-DARPinK27 was codon-optimized using an in-house algorithm and cloned into an in vitro transcription (IVT) vector. Using IVT, mRNA was synthesized with a co-transcriptional 5' CleanCap and complete uridine-to-pseudouridine substitutions. LNP Formulation

[0160] To formulate LNPs, an ethanol and an aqueous phase were mixed at a 1:3 volume ratio using a microfluidic device and pump 33 DS syringe pumps (Harvard Apparatus; Holliston, MA), as previously described. To prepare the ethanol phase, ionizable lipid, 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phos-phocholine (DOPC), or 1,2-dioleoyl- sn-glycero-3-phosphoethanol-amine (DOPE), lipid-anchored polyethylene glycol (PEG) (Avanti Polar Lipids; Birmingham, AL), and cholesterol (Sigma; St. Louis, MO) components were combined. For protein encapsulating LNPs, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was included in the ethanol phase as a fifth component, and the aqueous phase was prepared using 1x PBS, shifted to pH 5 with 150 mM sodium chloride. For mRNA encapsulating LNPs, only the original four lipid components were included in the ethanol phase, and the aqueous phase was prepared using 10mM citrate buffer. After mixing, LNPs were subsequently dialyzed against 1x PBS for 1 hour to remove ethanol. LNP Characterization

[0161] To determine hydrodynamic radius, LNPs were diluted 100x in 1x PBS in disposable cuvettes for dynamic light scattering (DLS) measurements on the Zetasizer Nano (Malvern Instruments; Malvern, UK). LNP size (Z-average diameter) and polydispersity index (PDI) are reported as the mean ± standard deviation of n = 3 measurements. To quantify surface zeta potential, LNPs were diluted 100x in water in DTA1070 zeta potential cuvettes (Malvern Panalytical, Malvern, UK) for measurement on the Zetasizer Nano instrument. mRNA concentration of mRNA encapsulating LNPs was determined using A260 / A280 absorbance measurements on a NanoQuant Plate (Tecan; Männedorf, Switzerland). For all LNP:proteinP-635623-PC formulations, the stated concentration refers to the total protein concentration (encapsulated and free) in solution based off the bioPROTAC input amount. Cryo-Electron Microscopy (cryo-EM) was performed by core personnel at the Beckman Center for Cryo-Electron Microscopy. LNP sample was concentrated 3-4x using Amicon Ultra Centrifugal Filters (10kDa MWCO, Millipore Sigma). Then, 3µL of concentrated LNPs were applied to a Quantifoil holey carbon grid which had been glow discharged. Grids were blotted, and plunge freezing was performed with liquid ethane using a Vitrobot Mark IV (Thermo Scientific). Imaging was performed on a Titan Krios (Thermo Scientific) equipped with a K3 Bioquantum. Protein Encapsulation Study

[0162] Proteins were labeled with a single C-terminal 5-carboxytetramethylrhodamine (5- TAMRA) dye via STEPL bioconjugation. Purified proteins were encapsulated into LNPs as described above and separated with columns packed with Sepharose CL-4B matrix (Cytiva). Fractions eluted by gravity chromatography were mixed with equal volumes of 0.1% Triton-X in black microwell plates and analyzed with a plate reader. Fractions corresponding to fluorescence peaks were pooled for DLS analysis. Plasmid DNA Transfection

[0163] For E3 screening assays, 293T cells were plated at 100,000 cells / well in 24-well plates overnight. The following day, pcDNA plasmids for bioPROTAC and GFP-KRAS expression were separately diluted in Opti-MEM reduced serum medium (Thermo Fisher Scientific). In total, 6 dilutions of pcDNA bioPROTAC and 4 dilutions of pcDNA GFP-KRAS were made ranging from 0ng / well to 500ng / well. Dilutions of the bioPROTAC and target plasmids were mixed pairwise to obtain 24 combinations of plasmids at 35µL final volume. Each of the 24 combinations was mixed with a separate solution comprising 2.5µL of Lipofectamine 2000 diluted into 35µL of Opti-MEM, and the resulting mixture was incubated at RT for 20 minutes to promote complexation. The DNA complexes were pipetted into wells, and plates was gently shaken to disperse the solution. After 8 hours, the culture media was replaced with fresh DMEM, and cells were grown for an additional 40 hours. At 48 hours post-transfection, cells were trypsinized and analyzed by flow cytometry.

[0164] For western blot analysis and validation of Ras-targeting bioPROTACs, co-transfection was performed in a 6-well format with 600,000293T cells seeded per well. In these experiments, 0.5µg of target plasmid and 2µg of bioPROTAC plasmid (except IpaH9.8-based degraders) were co-transfected with 5-10µL of Lipofectamine 2000. The amount of IpaH9.8-3G124 varied forP-635623-PC western blot analysis as indicated, and IpaH9.8-K27 / K27n3 was co-transfected at 0.25µg / well for flow cytometry. Cells were analyzed by western blotting or flow cytometry 24- or 48-hours post- transfection as indicated. Cytosolic Protein Delivery

[0165] In a typical protein delivery assay, cells were seeded overnight in either 6-well or 48-well plates such that they were 70-80% confluent at the time of transfection.

[0166] Lipofectamine-mediated delivery was performed in a 48-well format. First, stock proteins were diluted with Opti-MEM to achieve 20× the specified final treatment concentration in 10µL of Opti-MEM. Next, 2µL of Lipofectamine 2000 was diluted into 8µL of Opti-MEM, and the resulting mixture was thoroughly mixed with diluted protein by pipetting. The Lipofectamine / protein solution was allowed to complex for 15 minutes at RT, after which all 20µL of the mixture was added to wells containing 180µL of complete media. The corresponding lipid- free protein treatment samples were prepared by diluting stock proteins to 10x the indicated final concentration in 20µL of Opti-MEM.

[0167] LNP-mediated cytosolic protein delivery was performed in both 6-well and 48-well formats. In either format, LNP:protein was added directly to wells to achieve the desired final concentration. Flow Cytometry

[0168] At specified time points following either protein delivery or nucleic acid transfection, cells were detached from plates with 0.25% trypsin and pelleted at RT in a table-top centrifuge at 500 g. Cell pellets were resuspended in FACS buffer (1× PBS, 1% w / v BSA, 1 mM EDTA) and analyzed using a CytoFLEX flow cytometer (Beckman Coulter). At least 8000 cell-gated events were collected, and the geometric mean fluorescence intensity was calculated using FlowJo v10 software. For degradation assays involving 293T GFP-KRAS cells, data were normalized to untreated 293T GFP-KRAS cells and wild-type 293T cells. For split GFP delivery assays, the negative control sample was untreated GFP(1-10) cells, and the GFP-positive gate was defined such that only ~1% of the negative control sample would fall within that positive gate. Additionally, the geometric mean fluorescence intensity for each sample was divided by the mean fluorescence intensity of the negative control, and this ratio was taken as the fold-change MFI. siRNA-Mediated Knockdown

[0169] The day before transfection, 293T GFP-KRAS cells were plated overnight at 100,000P-635623-PC cells / well in a 24-well plate. Custom DsiRNA (IDT) designed to target KRAS were transfected into cells at a final concentration of 10nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific). An additional group was transfected with negative control DsiRNA (IDT) at a final concentration of 10nM. Transfections were performed according to manufacturer’s instructions. At indicated time points, cells were collected and analyzed by flow cytometry. The geometric mean fluorescence intensity was normalized to untreated and 293T wild-type samples, and the degradation rate of GFP-KRAS was estimated using a first-order decay model. mRNA Delivery

[0170] LNPs encapsulating bioPROTAC mRNA were added directly into cell culture wells to achieve the indicated final concentration. Western Blotting

[0171] Equal amounts of protein were boiled in sample loading buffer (928-40004, LI-COR Biosciences; Lincoln, NE) and resolved by SDS-PAGE with 4-12% Bolt Bis-Tris polyacrylamide gels (Thermo Fisher Scientific). Proteins were transferred onto PVDF membranes, blocked with Intercept TBS blocking buffer (LI-COR), and incubated with primary antibodies (diluted according to manufacturer’s recommendations) overnight at 4°C. The primary antibodies used in this study were: mouse anti-GFP (RT0265, Bio X Cell; Lebanon, NH), rabbit anti-Ras (3965, Cell Signaling Technology; Danvers, MA), rabbit anti-SAPK / JNK (9252, Cell Signaling Technology), rabbit anti-α-tubulin (2144, Cell Signaling Technology), rabbit anti-Erk1 / 2 (9102, Cell Signaling Technology), mouse anti-phospho-Erk1 / 2 (9106, Cell Signaling Technology), rabbit anti-Bcl-xL (2764, Cell Signaling Technology), and mouse anti-β-actin (3700, Cell Signaling Technology). Following primary antibody incubation, blots were incubated goat anti-rabbit 680RD (925-68071, LI-COR) and donkey anti-mouse 800CW (925-32212, LI-COR) IR-dye functionalized secondary antibodies (1:15,000 dilution). Membranes were scanned using an Odyssey M imaging system, and relative protein abundance was calculated by band densitometry using ImageJ software. Binding Assays

[0172] To measure the binding affinity of modified DARPins, biotinylated proteins were first produced by STEPL using a GGG-biotin peptide. Proteins were further processed as described above, serially diluted, and incubated overnight in black, 96-well streptavidin coated plates at 4°C. The following day, 50µL of either GFP (A42613, Thermo Fisher Scientific) or KRAS (156968, Abcam; Boston, MA) diluted to 5µg / mL was added to wells, and plates were gently shaken for 1.5P-635623-PC hours at RT. Then, either rabbit anti-GFP (600-401-215L, Rockland Immunochemicals, 1:10,000 dilution; Limerick, PA) or rabbit anti-Ras (3339, Cell Signaling Technology, 1:1000 dilution) was incubated in wells for 1 hour at RT. Finally, wells were incubated with a 1:4000 dilution of goat anti-Rb Ab-HRP for 1 hour RT (31460, Thermo Fisher Scientific). Between each incubation step, wells were washed 3x with 200µL of wash buffer (1x PBS, 0.05% Tween 20), and all proteins were diluted in Superblock T20 buffer (Thermo Fisher Scientific). To detect HRP, the QuantaRed substrate kit (15159, Thermo Fisher Scientific) was used according to manufacturer’s instructions. Wells not receiving GFP / KRAS were used for background subtraction, and data were fit with a 4- parameter logistic model using GraphPad Prism v10. LDH Cytotoxicity Assay

[0173] The day before treatment, 293T cells were plated at 30,000 cells / well in a 96 well plate in 80µL of complete media. The following day, LNPs were serially diluted in PBS to 5x the indicated final treatment concentration, and 20µL of the diluted LNPs were added to media to achieve the specified final treatment concentration. Cells were kept in the incubator at 37°C for 8 hours before cytotoxicity was measured using the Lactate Dehydrogenase (LDH) assay kit (CK12, Dojindo Molecular Technologies; Gaithersburg, MD) according to manufacturer’s instructions. Cell viability was normalized to dead and live controls in GraphPad Prism v10. Protein and LNP Stability

[0174] For protein stability studies, purified bioPROTACs were stored at either -80°C or 4°C in PBS for at least 4 weeks prior to cytosolic delivery and degradation analysis. To assess LNP stability, LNP:bioPROTAC was stored at 4°C, and degradation efficiency was tested every 2 days. In vitro Ubiquitination Assay

[0175] For each 25µL reaction, a master mix was made by combining 2.5µL of 10x reaction buffer (500mM HEPES, 500mM NaCl, 10mM TCEP), 1µL of ubiquitin (U-100H, R&D Systems; Minneapolis, MN), 2.5µL of MgATP solution (B-20, R&D Systems), 0.5µL of the human E1, UBE1 (E-304, R&D Systems), 1µL of the human E2, UbcH5b / UBE2D2 (E2-622, R&D Systems), 3µL of human KRAS (156968, Abcam), and 13.25µL of MQ H2O. To this master mix, 1.25µL of either 10µM bioPROTAC or 10µM control bioPROTAC was added and mixed well, initiating polyubiquitination. An additional negative control was included where 1.25µL of MQ H2O was added to the master mix instead of bioPROTAC. Reaction mixtures were incubated for 2 hours at 37°C and quenched with 25µL of 2x Tricine-SDS buffer. To detect ubiquitinated KRAS, at leastP-635623-PC 0.5µL of sample (15ng KRAS) was resolved by SDS-PAGE and probed by western blotting with an anti-KRAS antibody. To assay bioPROTAC autoubiquitination, proteins were first labeled with a single C-terminal TAMRA dye via STEPL, and in vitro ubiquitination was performed in the absence of target protein. Samples were resolved by SDS-PAGE, and ubiquitin-modified bioPROTACs were visualized by fluorescence imaging. Sample Preparation for Proteomic Analysis

[0176] Cells were harvested by scraping, pelleted by centrifugation, washed twice with ice-cold PBS, and snap frozen. Proteins were extracted by adding 200µL of ice-cold lysis buffer (8M urea, 75mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, protease inhibitor cocktail) to the cell pellet followed by 3 cycles of incubation on ice (5 minutes) and vortexing (10 seconds). Finally, samples were sonicated with an ultrasonic homogenizer and clarified by centrifugation at 20,000 g for 10 minutes, and the supernatant was collected for further analysis. Protein concentration was measured using a BCA Protein Assay Kit (A53227, Thermo Fisher Scientific), and equal amounts of protein (200µg) were taken from each sample. Samples were reduced using 5mM dithiothreitol (DTT) at 60°C for 30 minutes followed by cysteine alkylation using 20mM iodoacetamide (IAA) for 15 minutes in the dark. Samples were diluted to 1M Urea using 50mM Tris-HCl, pH 8.0, and trypsin digestion was performed with modified sequencing-grade trypsin (Promega, Madison, WI) at a 1:30 trypsin-to-protein ratio overnight at 37°C. Basic Reverse Phase Liquid Chromatographic (bRPLC) Fractionation of Digested Peptides and C18 Clean-Up

[0177] Peptide fractionation was carried out using a C-18 StageTip protocol eluting with a high pH mobile phase (50mM Triethylammonium bicarbonate pH 8.5 (TEABC)). Briefly, the C-18 material was packed into 200μL pipette tips, activated with 100% acetonitrile (ACN), and equilibrated with 50mM TEABC. Peptides were resuspended in 50mM TEABC and loaded onto the C-18 StageTips column. Samples were passed twice through the column, followed by washing with 50mM TEABC. Finally, samples were eluted with increasing concentrations of ACN (5-50%) in TEABC to yield a total of 8 fractions and combined pairwise (1+5, 2+6, 3+7, 4+8) to give a total of 4 fractions. Peptide desalting was carried out with the C-18 StageTip method. The C-18 material was packed into 200μl pipette tips, activated with 100% ACN, and equilibrated with 0.1% formic acid in ACN. Fractionated peptide samples were resuspended in 0.1% formic acid and loaded onto the C-18 StageTip. Samples were passed through the column twice, followed byP-635623-PC washing with 0.1% aqueous formic acid in ACN and elution with 40% ACN in aqueous 0.1% formic acid. The eluent was dried and stored at −20°C until LC-MS / MS analysis. Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS)

[0178] Cleaned peptides were analyzed on Thermo Scientific Orbitrap Exploris 240 mass spectrometer interfaced with Thermo Scientific UltiMate 3000 HPLC and UHPLC Systems. Peptide digests were reconstituted in 0.1% formic acid and were separated on an analytical column (75 µm × 15 cm) at a flow rate of 300nL / min using an increasing gradient of solvent B (0.1% formic acid in 100% acetonitrile). The total run time was set to 120 min. The mass spectrometer was operated in data-dependent acquisition mode. A survey full scan MS (m / z 400–1600) was acquired in the Orbitrap with a resolution of 6000 normalized AGC target of 300%. Data were acquired in topN with 20 dependent scans. Ions were fragmented using 37% normalized collision energy and detected at a mass resolution of 1500. Dynamic exclusion was set for 8s with a 10ppm mass window. Proteomic Data Analysis

[0179] MS / MS searches were performed with SEQUEST against the Uniprot database for human proteins supplemented with bioPROTAC sequences using Proteome Discoverer (Version 3.0, Thermo Fisher Scientific; Bremen, Germany). The workflow included Spectrum files, Spectrum selector, SEQUEST search nodes, target decoy PSM validator, peptide validator, event detector, precursor quantifier. Data was searched in label free quantification mode using unique peptides for quantification. Oxidation of methionine and N-terminal protein acetylation were used as dynamic modifications and carbamidomethylation of cysteine was set as a static modification. MS and MS / MS mass tolerances were set to 10 ppm and 0.05 Da, respectively. A maximum of two missed cleavage was allowed. Target-decoy database searches used for calculation of false discovery rate (FDR) and for peptide identification FDR were set at 1%. Feature mapper and precursor ion quantifier were used for label-free quantification. Custom R scripts were used for downstream data analyses and visualizations. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD049388. Size Exclusion Chromatography for Protein Interaction Analysis

[0180] Recombinant eGFP and binding partners were diluted with PBS to a final concentration of 10µM per protein in a final volume of 200µL. Samples were incubated at RT for 10 minutes andP-635623-PC centrifuged at 12,000 g to remove aggregates. Protein mixtures were injected into an ӒKTA pure chromatography system (Cytiva) with a Superdex 200 Increase 10 / 300 GL column installed. Eluted fractions were pooled and analyzed by SDS-PAGE. Fluorescence Microscopy of HeLa and U2OS

[0181] HeLa and U2OS cells expressing GFP-fusion proteins were either left untreated or treated with 100nM K1:bioPROTAC targeting GFP. After 7.5 hours, cells were stained with Hoechst 33342 at 1µg / mL for 20 minutes. After nuclei staining, media was aspirated and replaced with FluoroBrite DMEM (A1896701, Thermo Fisher Scientific). Cells were imaged using a Nikon Ti2- E microscope equipped with a Yokagawa CSU-W2 spinning disk, and an sCMOS camera (Photometrics). Images were acquired using a 20× air objective and the 405nm and 488nm laser lines for Hoechst and GFP respectively. Images were background subtracted and equalized with ImageJ software. Time-Course Confocal Microscopy

[0182] 293T GFP-KRAS / iRFP-CaaX cells were seeded overnight at 100,000 cells / well in glass 96-well plates treated with 10µg / mL human plasma fibronectin (FC010, Thermo Fisher Scientific). The following day, LNPs were pipetted directly into wells, and cells were imaged every 20 minutes for 12 hours using a 40× air objective. Throughout the duration of the experiment, temperature and CO2 were maintained at 37°C and 5% respectively using an environmental chamber (Okolab; Sewickley, PA). Images were acquired using a Nikon Ti2-E microscope equipped with a Yokagawa CSU-W1 spinning disk, 405 / 488 / 561 / 640 nm laser lines, and an ORCA-fusionBT digital camera C15440 (Hamamatsu). Time-Course Image Analysis

[0183] Image pre-processing was performed using ImageJ with the MorphoLibJ plugin (imagej.net / plugins / morpholibj). First, GFP and RFP channel images were equalized, and GFP channel images were further background subtracted using the rolling-ball algorithm. To segment individual cells, a morphological closing operation (octagon element, radius = 15 pixels) was applied to RFP channel images, and a watershed algorithm was implemented (tolerance = 200). The resulting catchment basins were extracted and loaded into CellProfiler85as cell objects, while processed GFP-channel images were uploaded as grayscale images. Finally, a custom pipeline was used to measure the integrated and membrane-associated fluorescence intensity of individual cells. Between 400-500 single cells were analyzed at each time point and their calculated fluorescenceP-635623-PC intensities were averaged and normalized to fluorescence intensity at t = 0. Real-Time Cell Viability Assay

[0184] Cells were plated in a 96-well E-plate (Agilent; Santa Clara, CA) and allowed to attach overnight. The following day LNPs were added to wells to achieve the indicated final protein or mRNA concentrations, and proliferation was monitored using the xCELLigence Real-Time Cell Analysis system (Agilent). The cell index, an arbitrary unit of confluence was normalized to untreated controls for analysis. Protein Structural Model

[0185] The structure of the Ras-targeting IpaH9.8-K27 bioPROTAC was predicted by AlphaFold. The resulting model was then aligned with IpaH9.8 NEL (PDB ID: 6LOL) and DARPinK27 (PDB ID: 5O2S) with PyMol. Statistics

[0186] Multiple batches of proteins and LNPs were used throughout this study. Two-tailed t tests and ANOVA were used for data analysis. When appropriate, multiple-comparisons testing was performed, and Bonferroni correction was applied. All statistical analyses were performed using GraphPad Prism v10. EXAMPLE 2 Lipid-Mediated Intracellular Delivery of Recombinant bioPROTACs for the Rapid Degradation of Undruggable Proteins and Cancer Therapy Results Identifying active N-terminal E3 domains for bioPROTAC construction

[0187] A modular bioPROTAC format was established by first screening E3 ligases for degradation activity when fused to the N-terminus of a target-specific DARPin. In total, five E3 domains reported to have high activity were chosen: CHIP, SKP2, SOCS2, SPOP, IpaH9.8 (Figure 2A). For all E3 proteins tested, only their degradation domains capable of recruiting E3 complex proteins or E2 conjugating enzymes were used, and the native substrate binding portions were not included in the bioPROTAC designs. Of note, the IpaH9.8 domain used in this study is not a true mammalian E3 ligase. Instead, the protein is a novel E3 ligase (NEL) derived from Shigella flexneri virulence factors, and its native role is to degrade NEMO and suppress the NF- κB inflammatory response. Nonetheless, it was included IpaH9.8 for its ability to recruit host E2P-635623-PC enzymes for efficient polyubiquitination. With the exception of SKP2, all E3 ligases tested have their natural substrate recognition domain at the N-terminus, and previous groups simply replaced this sequence with desired scaffolds. In contrast to this, the inventors chose, instead, to install E3 domains at the N-terminus regardless of their natural orientation, as it was previously found that C-terminal placement of polyaspartic acid ApP tags (D25, D30) preserved both expression yields and binding affinity of DARPin scaffolds. Moreover, fusion protein designs where the ApP was placed between the binder and E3 were not considered in this screen. The GFP-binding DARPin 3G124 was used as the targeting domain to allow for a facile fluorescent read-out of successful degradation, and the candidate E3-DARPin formatted bioPROTACs were cloned into pcDNA vectors for characterization.

[0188] To benchmark bioPROTAC performance, 293T cells were co-transfected with pcDNA plasmids encoding GFP-KRAS and one of the five bioPROTACs at various bioPROTAC:target ratios (Figure 2B). An additional plasmid encoding only the anti-GFP DARPin was also included as a negative control. After 48 hours, cells were analyzed by flow cytometry to assess GFP degradation levels (Figure 2C-2H). No reduction in GFP was observed in cells co-transfected with 3G124 and GFP-KRAS indicating binding alone did not result in degradation. On the contrary, an increase in GFP levels was seen when 3G124 was transfected suggesting DARPin-mediated stabilization of GFP-KRAS. Dose-dependent degradation was observed with SKP2- and SOC2- based bioPROTACs with SOCS2 exhibiting higher activity. Notably, CHIP failed to induce target elimination at any dose, despite its usage in multiple reported bioPROTAC designs. Finally, both SPOP-3G124 and IpaH9.8-3G124 exhibited the highest activity with sharp GFP reductions even at low bioPROTAC amounts. When transfected with the 500ng of GFP-KRAS, just 62.5ng of SPOP and IpaH9.8 bioPROTAC plasmids resulted in 70% and >90% degradation respectively. GFP-KRAS depletion was further validated by Western blotting. Consistent with flow cytometry results, SPOP degraded GFP-KRAS efficiently, while SKP2 and SOCS2 displayed weaker activity (Figure 2I). Interestingly, potent IpaH9.8-induced degradation was observed at low bioPROTAC:target ratios but not at high bioPROTAC levels (Figure 2J) indicating the existence of a hook effect. This phenomenon occurs when PROTAC treatment preferentially drives the formation of POI:PROTAC and PROTAC:E3 binary complexes rather than ternary complexes needed for degradation resulting in a paradoxical reduction in degradation efficiency at higher PROTAC concentrations. As IpaH9.8 binds directly to E2 conjugating enzymes, the hook effect observed here would occur through the formation of E2:bioPROTAC binary complexes rather thanP-635623-PC through E3 saturation. Based on this screen, both SPOP and IpaH9.8 were chosen as lead E3s, and their developability was established by successful purification as DARPin-fusion proteins that retained substrate binding capabilities (Figs. 10A-10F). bioPROTAC delivery with commercially-available transfection reagents

[0189] Next, 3G124 was replaced with DARPinK2754(K27) to redirect degraders towards undruggable Ras proteins (Figures 3A, 3B). As with 3G124 bioPROTACs, transfection of 293T cells with K27-based bioPROTACs produced potent degradation of co-transfected GFP-KRAS. Here, target destruction is achieved by binding to the KRAS handle. To rule out non-specific degradation, bioPROTACs containing a null K27 mutant with abrogated binding (K27n3) were also tested and were not observed to reduce GFP levels (Figures 3C, 3D). After verifying bioPROTAC functionality by transient DNA transfection, it was investigated whether bioPROTACs would display similar activity as exogenously-delivered purified proteins. The complete bioPROTAC format includes a target-specific scaffold fused with an N-terminal E3 domain (SPOP or IpaH9.8) and a C-terminal D25 ApP tag. A panel of control proteins was purified to dissect the necessary components of a cytosolically-delivered bioPROTAC system (Figure 3E, Figs.11A-11H). Specifically, a binder-only control (no E3 domain), binding-deficient controls (K27n3), and non-charged (no ApP) variants were cloned and purified. For all proteins, a GFP s11 peptide was included as a reporter of intracellular delivery. The small, 16 amino acid tag does not interfere with protein function and can be used for stringent detection of cytosolic delivery when transfected into cells stably expressing the complementary GFP(1-10) fragment.

[0190] Either SPOP-K27-D25-s11 or IpaH9.8-K27-D25-s11 was complexed with off-the-shelf cationic Lipofectamine 2000 reagent and delivered into 293T cells stably expressing GFP-KRAS (Figure 3F). In agreement with DNA transfection experiments, successful degradation (leftward shift in flow histograms) was observed following intracellular delivery of IpaH9.8-fused bioPROTACs (Figure 3G). However, GFP depletion was not detected following treatment with Lipofectamine-complexed SPOP-K27-D25-s11. It is unclear why the purified SPOP-based bioPROTAC was unable to deplete GFP-KRAS, as they were able to bind to targets (Figures 10C, 10D). Moreover, chromatograms of SPOP bioPROTACs indicated oligomerization of the E3 ligase (Figures 10C, 11C-11E), suggesting proper protein folding. Thus, it was concluded that the delivery efficiency and / or potency of SPOP-K27-D25-s11 is low and proceeded with IpaH9.8- based degraders for further development.P-635623-PC

[0191] Next, the roles of IpaH9.8 and ApP domains on bioPROTAC affinity, intracellular delivery, and target degradation were evaluated. First, binding of the complete bioPROTAC (IpaH9.8-K27-D25-s11) against KRAS was assayed, and the affinity was found to be comparable to that of K27-s11 against KRAS (Figures 12A-12B). Therefore, it was concluded that placement of the DARPin between E3 and ApP did not interfere with substrate recognition. To determine if IpaH9.8 and the D25 ApP domains were necessary for degrader functionality, complete Ras- targeting bioPROTACs and a series of controls were either complexed with Lipofectamine 2000 prior to delivery or directly added to 293T GFP-KRAS culture media (Figure 4A). For each condition, 500nM of protein was used, and degradation was quantified by flow cytometry 8 hours after treatment. Cytosolic delivery of Lipofectamine-complexed IpaH9.8-K27-D25-s11 resulted in a 46% reduction of GFP-KRAS levels, whereas control proteins lacking E3, binding ability, or ApP could not deplete GFP-KRAS levels. Similarly, the complete bioPROTAC on its own produced no degradation, highlighting the membrane impermeability of these molecules without the aid of a delivery agent.

[0192] To assay cytosolic delivery efficiency of bioPROTACs, 293T GFP(1-10) reporter cells were treated with the same panel of proteins with or without Lipofectamine (Figures 4B, 4C). As expected, only proteins fused with D25 ApPs could complex with Lipofectamine and reach the cytosol. Successful cytosolic delivery was confirmed by increases both in GFP-positivity, a measure of cell transfection efficiency, and in geometric mean fluorescence intensity (MFI), a metric for the amount of protein delivered to cells. The non-binding IpaH9.8-K27n3-D25-s11 control could also be delivered into cells with the same efficiency as active bioPROTAC but was unable to degrade targets. It was confirmed that this was due to the protein’s inability to polyubiquitinate KRAS (Figure 13). Hence, it was shown that the E3 ligase, target-specific binder, and negatively-charged ApP are fundamental requirements for protein degradation in this bioPROTAC platform.

[0193] Since Lipofectamine is a readily-available transfection reagent, Lipofectamine-mediated bioPROTAC transfection was further characterized for utility as a research tool. Following incubation with Lipofectamine:bioPROTAC, a dose-dependent decrease in GFP levels was observed in 293T GFP-KRAS, and at 8 hours post-treatment, a maximum degradation efficiency of 45% was reached (Figure 4D). This coincided with a peak delivery efficiency of ~22% and a 1.85-fold increase in MFI compared to untreated cells (Figures 4E, 4F). Both degradation and delivery efficiencies were maximized at 125nM bioPROTAC complexed with 2µL ofP-635623-PC Lipofectamine. Furthermore, both degradation efficiency and delivery efficiency were found to be time dependent (Figures 14A-14C). Lipofectamine-delivered bioPROTACs were also compared to commonly-used RNAi methods, and 293T GFP-KRAS cells were transfected with two different KRAS-targeting siRNA using Lipofectamine RNAiMAX (Figure 14D). In this head-to-head comparison, bioPROTACs displayed greater than 3 times faster degradation kinetics than siRNA (Figure 14E).

[0194] Taken together, purified ApP-tagged bioPROTACs could serve as a powerful research tool when paired with easily-accessible cationic transfection reagents and would facilitate biological interrogation at much shorter time scales without the need to genetically modify cells. It also was shown that this knockdown approach circumvents one of the intrinsic limitations of siRNA (slow kinetics) for potential therapeutic applications. As another advantage over RNA, the stability of purified bioPROTACs under simple storage conditions was tested and verified that they displayed no loss in activity after four weeks when stored in PBS at 4°C (Figure 15). Identifying an optimal LNP formulation for intracellular delivery of bioPROTAC protein

[0195] It is important to note that despite improved target knockdown compared to siRNA, complete degradation was not achieved by Lipofectamine-mediated transfection of bioPROTACs. It was hypothesized that this incomplete elimination by Ras-targeting degraders was due to low Lipofectamine transfection efficiency. To improve intracellular protein delivery, LNPs were explored for bioPROTAC encapsulation, as they exhibit high drug loading capacity and promote endosomal escape for enhanced cytosolic. To develop LNPs for bioPROTAC encapsulation, three base formulations were used (Table 1 supra). Proteins were formulated into LNPs by microfluidic mixing of a bioPROTAC-containing aqueous phase and lipid- / excipient-containing ethanol phase (Figure 5A). All three formulations performed better than Lipofectamine 2000, validating our initial reasoning for using LNPs. Excitingly, treatment with the K1 formulation led to 95% GFP- KRAS elimination at a 200nM bioPROTAC dose (Figures 5B, 5C). Interestingly, while the B6 formulation was previously optimized for DARPin-ApP delivery, it only displayed a degradation efficiency of 60%. To study how LNP composition contributed to LNP:bioPROTAC formation and subsequent degradation efficiency, a library of LNP formulations was generated based off of the K1 formulation which includes C12-200 as the ionizable lipid (Table 2 supra). Formulations incorporating 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the helper lipid exhibited the highest degradation efficiency (Figure 16A). Ultimately, the K1 formulation was chosen as the lead LNP due to its functional potency and physical properties. The formulationP-635623-PC retained high degradation efficiency up to one week following formulation (Figure 16B), had a relatively small hydrodynamic diameter of 167nm, and exhibited near-complete encapsulation of protein cargo (Figures 16C-16E, Table 3 supra).

[0196] The degradation dose response of K1:bioPROTAC was investigated in 293T GFP-KRAS cells by flow cytometry, and a half-maximal degradation (DC50) of 19.5nM was calculated at 8- hours post-treatment (Figure 5D). The acute cytotoxicity of LNP:bioPROTAC in 293T cells was determined using a lactate dehydrogenase (LDH) assay, and cell viability was found to be >90% at protein concentrations up to 100nM (Figure 5E). The increased target clearance by LNP:bioPROTAC was accompanied by a dramatic improvement in delivery efficiency. After treatment with 200nM LNP-delivered bioPROTAC, 49% of 293T GFP(1-10) were GFP-positive, representing a two-fold increase in cytosolic delivery efficiency over Lipofectamine (Figure 5F). The amount of protein delivered was commensurate, with LNP:bioPROTAC achieving a 3-fold change in MFI over the control group (Figure 5G). For both LNP and Lipofectamine delivery approaches, it was noticed that the degradation efficiency was greater than the delivery efficiency. This can be attributed to the non-stoichiometric mechanism of bioPROTACs in which one degrader molecule can catalyze the destruction of many target molecules. Moreover, the limit of detection for split GFP assays is in the low nanomolar range36, meaning split GFP may lack sufficient sensitivity to reflect highly-active degradation activity.

[0197] Next, it was tested to find out if K1:bioPROTAC could degrade endogenous Ras as opposed to GFP-KRAS fusion proteins used in characterization studies. To this end, wild-type (WT) 293T cells were treated with LNPs encapsulating either K27-D25-s11, IpaH9.8-K27-D25- s11, or IpaH9.8-K27n3-D25-s11. Lysates from treated cells were analyzed by western blotting and probed with a pan-Ras primary antibody (Figure 5H). Closely mirroring flow cytometry results, incubation with 100nM K1:bioPROTAC for 8 hours resulted in up to 80% endogenous Ras degradation as calculated by band densitometry (Figure 5I). In accordance with the proposed mechanism of action, neither B6:K27-D25-s11 nor K1:bioPROTAC(null) led to reductions in Ras levels owing to a lack of E3 activity and binding affinity respectively, and the bioPROTAC protein alone did not induce target degradation due its inability to enter cells without a delivery vehicle. A panel of cancer cells was also treated with LNP:bioPROTAC to validate the generalizability of our protein degrader in multiple cell types. A similar pattern of degradation was observed in HCT116 colorectal cancer cells, A549 lung cancer cells, and HT1080 fibrosarcoma cells. On average, the Ras degradation efficiency in these cell lines ranged from approximately 40% to 55%P-635623-PC (Figures 17A-17F). Target degradation kinetics with LNP-delivered bioPROTAC

[0198] To study the degradation kinetics of LNP-delivered bioPROTACs, 293T GFP-KRAS cells were further engineered to express iRFP-CaaX, a fluorescent membrane marker. These dual- reporter cells enabled simultaneous degradation analysis and live-cell tracking via an orthogonal mask. As a direct comparison to direct protein delivery, a treatment group receiving LNP- encapsulated mRNA encoding IpaH9.8-K27 (bioPROTACmRNA) was added. Cells were incubated with LNPs at dosages producing maximum degradation (Figure 5D, Figure 18), and GFP was monitored for 12 hours. As expected, cell-only controls exhibited no changes in GFP fluorescence intensity (Figures 6A, 6D,). Meanwhile, both LNP-mediated bioPROTAC protein delivery (Figures 6B, 6E,) and bioPROTAC mRNA delivery (Figures 6C, 6F) resulted in rapid and robust GFP clearance, reaching maximum degradation efficiency in 5 hours. The target half-life following protein delivery was calculated to be 97 minutes, while the half-life for mRNA was calculated to be 146 minutes. Treatment with either null bioPROTAC or binder only controls did not lead to degradation and actually resulted in an apparent stabilization of the target protein (Figures 19A-19D).

[0199] For many therapeutic applications, prolonged degradation is desirable, so it is critical to understand the duration of bioPROTAC activity following LNP-mediated delivery. Flow cytometry was used to analyze LNP-treated cells at various time points up to 48 hours post- delivery with either bioPROTACproteinor bioPROTACmRNA(Figure 6G). At both low and high doses, proteins produced sharp GFP depletion within 4 hours. The maximum degradation efficiency was nearly identical for both modalities, reaching up to ~95% target reduction by 8 hours. Recovery of GFP-KRAS levels was similarly comparable between protein and RNA. Under treatment washout conditions (LNP-containing media replaced with fresh media at 4 hours), complete recovery was observed by 48 hours. Conversely, sustained presence of LNPs in solution (no washout) resulted in only 30-40% recovery at 48 hours. Global proteome response to bioPROTAC treatment

[0200] To understand how bioPROTACs were affecting protein levels globally, shotgun proteomics were performed following degrader delivery. Cells were either left untreated, treated with K1:Ras bioPROTAC protein, or transfected with LNP:bioPROTAC mRNA. A group receiving K1:null bioPROTAC protein was also included to examine the specificity of theP-635623-PC degraders. Cells were treated for 8 hours, and extracted proteins were subjected to tandem mass spectrometry (MS). Label-free quantification resulted in 4752 uniquely-identified proteins common to all treatment groups. After filtering proteins with PSM > 24, a final dataset of 3827 proteins was used for downstream analysis. Proteins were considered differentially-expressed if the log2 fold-change (log2fc) between treated and untreated groups was either ≤ -1 or ≥ 1 and the p-value < 0.05. In both mRNA bioPROTAC and protein bioPROTAC groups, NRAS and KRAS abundance was lower compared to no treatment, although KRAS did not meet the log2fc threshold in the protein-treated group (Figures 7A, 7B). Knockdown of NRAS and KRAS was not observed in cells treated with null bioPROTAC (Figure 7C). These results matched the expected depletion pattern in samples treated simultaneously and analyzed by western blotting (Figure 20A). In addition to NRAS, also identified were several proteins significantly downregulated in active bioPROTAC treatment groups but in the null bioPROTAC treatment group (Figure 7D). Amongst these, members of the trimeric G-protein family: GNAI1, GNAI2, and GNAI3 were found to be depleted by both bioPROTAC protein and mRNA treatment. These proteins are structurally homologous to Ras-family proteins, containing P-loop and G-Box motifs also found near the DARPinK27-KRAS binding site. Thus, it was suspect guanine nucleotide-binding protein G(i) subunit alpha is a bona-fide off-target for DARPinK27. The relationship between the remaining proteins is less clear. However, all of them are membrane-associated. Furthermore, CHMP4A, MYO1B, and MYO1D are implicated in endosomal trafficking processes. Specifically, CHMP4A is known to facilitate lysosomal degradation of ubiquitinated membrane proteins, and ubiquitination-dependent lysosomal trafficking has been documented for other surface receptors. These data raise the possibility that other degradation pathways including endosomal-lysosomal sorting contribute to bioPROTAC-mediated target depletion, at least in the case for membrane- associated targets.

[0201] It also was noticed that a high percentage of shared down proteins between mRNA and null bioPROTAC that were not identified in the protein bioPROTAC group (Figure 7D). To better understand these downregulated proteins, Gene Ontology (GO) biological process (BP) enrichment analysis was performed and it was found that many members of this group mapped to RNA splicing machinery (Figure 20B). Prior reports have found that IpaH9.8 binds to and inhibits U2AF splicing factors18 other U2AF-interacting proteins were downregulated, suggesting co- regulation by the IpaH9.8 domain (Figure 20C). Although our bioPROTAC does not include the IpaH9.8 substrate-recognition domain, U2AF binding was found— counterintuitively—to occurP-635623-PC through the IpaH9.8 C-terminal domain62. Interestingly, the active bioPROTAC protein induced non-significant decreases in U2AF despite also having the IpaH9.8 catalytic module. This mitigated off-target effect could be because the protein’s targeting domain redirects its activity away from U2AF. In addition, its intracellular concentration at 8 hours could be lower compared to mRNA-expressed bioPROTAC.

[0202] Despite effectively degrading NRAS and KRAS, mRNA treatment led to substantially more differentially-regulated proteins compared to bioPROTAC protein delivery. Some of these proteins were assigned to mRNA processing pathways via GO enrichment analysis (Figure 7E) and are likely cellular responses to exogenous mRNA delivery. Molecular function enrichment analysis was performed on significant mRNA treatment knockdowns, and excluding Ras, an additional 12 GTPases that were degraded by mRNA bioPROTAC were uncovered. This finding was further investigated and the fold-change of all GTPases found to be downregulated either in mRNA or protein bioPROTAC treatment groups were compared. With the exception of RhoB, both protein and mRNA-mediated degradation of Ras and Ras-like proteins were correlated (Figure 7F). Interestingly, a subset of GTPases with little homology to Ras-family proteins was identified, including α- and β-tubulins, that were strongly downregulated by mRNA bioPROTAC but not by recombinant protein bioPROTACs, indicating plausible modality-specific degradation off-targets. Moreover, transfection with mRNA produced many additional downregulated proteins that were not accounted for by pathway or molecular function enrichment. Taken together, these results demonstrate that protein knockdown profiles can differ greatly depending on whether bioPROTACs are delivered as proteins or expressed from mRNA. Mechanistic exploration of bioPROTAC target- and self-degradation

[0203] Autoubiquitination and self-degradation are major concerns with our bioPROTAC design. This is because the NEL domain used in our constructs lacks the native auto-inhibitory mechanism present in full-length IpaH9.8 critical to preventing self-targeting. To investigate the fate and degradation mechanism of our bioPROTACs, a catalytically-dead variant was produced by mutating the catalytic cysteine residue in IpaH9.8 NEL to alanine. IpaH9.8C337A-K27-D25-s11 was purified and its inactivation via in vitro ubiquitination assays was confirmed (Figure 21A-21C). t was found that this inactivation was not due to changes in binding affinity (Figure 21D). Furthermore, wild-type bioPROTACs were prone to autoubiquitination (Figure 21E), and this self-targeting was completely abolished in the C337A mutant. Upon delivery into GFP(1-10)P-635623-PC reporter cells, the IpaH9.8C337Avariant displayed higher apparent transfection efficiency compared to bioPROTACs fused to IpaH9.8WT(Figures 22A-22C). This increased GFP signal indicates higher levels of bioPROTAC in the cytosol. As there is only a single amino acid difference between the two proteins, this discrepancy should not stem from differences in cargo encapsulation or delivery efficiencies. Instead, it was proposed that the bioPROTACs self-ubiquitinate upon intracellular delivery, reducing the cytosolic pool of degrader proteins. Addition of MG-132, a peptide proteasome inhibitor, to culture media partially rescued degradation of GFP-KRAS and completely rescued endogenous Ras (Figures 22D-22E). Taken together, these data indicate that bioPROTAC-mediated target degradation does occur via the ubiquitin-proteasome pathway as expected. However, the incomplete rescue of GFP-KRAS points to possible contributions from alternative degradation pathways.

[0204] To determine if the lysosome plays a role in bioPROTAC-mediated degradation, cells were treated with chloroquine and bafilomycin A1, either alone or in combination with MG-132. Like MG-132, it was found that chloroquine alone partially rescued GFP-KRAS degradation following bioPROTAC (IpaH9.8WT-K27-D25-s11) delivery. While bafilomycin A1 appears to rescue degradation of GFP-KRAS, a parallel split GFP complementation assay revealed that bioPROTAC protein delivery was almost completely suppressed (Figure 22F). This finding is in agreement with other reports that bafilomycin A1 prevents LNP endosomal escape by inhibiting lysosomal acidification64,65. On the other hand, chloroquine did not block LNP delivery, thus implicating the lysosome in IpaH9.8 bioPROTAC-mediated degradation of GFP-KRAS. Moreover, cells treated with both MG-132 and chloroquine displayed higher split GFP complementation (6-fold MFI increase) compared to bioPROTAC delivery alone (~3-fold MFI increase), suggesting that a combination of proteasomal and lysosomal pathways partake in bioPROTAC self-destruction. Chloroquine did not rescue degradation in 293T WT cells following bioPROTAC treatment, further supporting a UPS-dominated degradation mechanism for endogenous Ras (Figure 22G). Taken together, these data provide evidence that multiple pathways including proteasomal and lysosomal degradation can contribute to bioPROTAC-mediated degradation. The relative contributions of each pathway are likely to be influenced by the specific bioPROTAC / target pair as well as target expression levels, among other factors. Modularity of our bioPROTAC format

[0205] To examine the ability of our bioPROTAC platform to degrade diverse targets, K27 wasP-635623-PC replaced with alternate DARPin sequences, redirecting their activity towards other endogenous substrates (Figure 8A). DARPins that bind extracellular signal-regulated kinase 1 / 2 (Erk 1 / 2), c- Jun N-terminal kinases (Jnk), or B-cell lymphoma-extra large (Bcl-xL) were chosen, as these proteins are promising therapeutic candidates being pursued for chemical inhibition. All four alternate bioPROTACs could be purified with good yield (Figure 8B). The bioPROTACs were encapsulated in LNPs using the K1 formulation and delivered to either 293T or A549 cells. Clear depletion of Jnk and Erk bands were observed by western blotting following treatment with J1 / 2_2_25 and EpE89 respectively (Figure 8C, lanes 3 and 4). By contrast J1 / 2_2_3 and 012_F12 failed to noticeably degrade their respective targets: Jnk and Bcl-xL (Figure 8C, lanes 2 and 5). Notably, while both J1 / 2_2_25 and J1 / 2_2_3 are N2C-formatted DARPins identified from separate screens against Jnk, they produced different degradation outcomes. When degradation was observed, it was highly specific, as each unique bioPROTAC degraded only their intended targets while sparing the other surveyed proteins. This success rate for alternate bioPROTACs was achieved with no additional engineering of the bioPROTAC scaffold, and alternate degraders were easily produced by “plug-and-play” cloning. Thus, in agreement with previous reports of cell- expressed bioPROTACs, it was concluded that our purified, ApP-tagged degrader format is modular and exhibits a high degree of design flexibility.

[0206] Whether the bioPROTAC format could degrade targets localized to various intracellular compartments was investigated. Both Ras- and GFP-targeting degraders already demonstrated potent activity against GFP-KRAS and endogenous Ras, both of which associate with the inner leaflet of the plasma membrane. In addition, the GFP-targeting bioPROTAC (IpaH9.8-3G124- D25-s11) was tested against a panel of cell lines stably expressing GFP-fusions. Using the K1 formulation for cytosolic delivery, bioPROTACs effectively eliminated GFP localized to the nucleus (GFP-MIS12, GFP-TRF1), cytosol (GFP-SPC25) (GFP-MITO). Representative fluorescent images showed near-complete degradation just 8 hours after LNP incubation (Figure 8D). This result was further confirmed by flow cytometry analysis. Again, GFP-targeting bioPROTACs completely silenced fluorescence signal, whereas Ras-targeting bioPROTACs, used as a negative control, had no effect on target levels (Figure 8E). Inhibition of pancreatic tumor cells with a Ras-degrading bioPROTAC

[0207] To demonstrate a potential therapeutic application of LNP: bioPROTACs, the antiproliferative effects of Ras-degrading bioPROTACs when delivered into MIA PaCa-2, aP-635623-PC PDAC line harboring a KRAS G12C driver mutation, were examined. First, it was confirmed that Ras-targeting bioPROTACs were functional once delivered into MIA PaCa-2. Cells were incubated with K1 LNP formulations of either active or control bioPROTAC proteins containing either the C337A mutation and / or null K27n3 DARPin. Following 8-hour protein delivery, cell lysates were analyzed by Western blotting (Figure 23C). Following LNP treatment, Ras was depleted in a dose-dependent manner with IpaH9.8-K27-D25-s11 protein delivery, but not in any of the other treatment groups (Figures 9B, 9A, 9F, 9G). Also probed was phosphorylated Erk (pErk), a key effector in the canonical MAPK signaling pathway and observed its depletion in tandem with Ras degradation. Despite prior confirmation that IpaH9.8C337A-K27-D25-s11 retained its ability to bind Ras (Figure 21D), it was not able to block Ras signaling in the MIA PaCa-2 cell line (Figure 9F, Figure 23C). Next, cells were treated with the same proteins formulated as K1 LNPs, and proliferation was monitored using an impedance-based confluency assay. At 24 hours post-treatment, K1:IpaH9.8-K27-D25-s11 resulted in a 46% reduction in growth. These effects were statistically significant when compared to control proteins lacking binding and / or degradation ability (Figure 9H). As the IpaH9.8-K27 bioPROTAC was the only protein able to block canonical MAPK signaling, our results indicate that anti-proliferative effects were due to Ras targeting. These results were compared against bioPROTAC mRNA treatment, which displayed comparable Ras degradation, pErk reduction, and viability inhibition at mRNA doses between 75-150ng / mL (Figures 9C, 9I).

[0208] Despite promising growth inhibition with K1:bioPROTAC, some viability loss in MIA PaCa-2 cells treated with K1-formulated null bioPROTAC at higher doses was noticed, indicating non-specific toxicity. To alleviate toxicity arising from the particles themselves, variations of the K1 formulations substituting the C12-200 lipid with a series of commercially-available and previously-published ionizable lipids (Figure 24A-24B) were screened. It was found that LNPs incorporating SM-102 retained anti-proliferative effects, exhibiting ~70% growth inhibition while reducing non-specific toxicity to 18% at a 140nM protein dose (Figure 24E). It was confirmed that all bioPROTAC-containing LNPs degraded endogenous Ras (Figure 24I). Altogether, these results show that LNP-delivered recombinant bioPROTACs can exert therapeutic effects by degrading oncogenic proteins. By tuning LNP formulations, it is possible to obtain therapeutic profiles comparable to mRNA delivery (Figure 24J), a modality that has been readily adopted by the bioPROTAC field.P-635623-PC Discussion

[0209] In summary, the inventors have developed a modular recombinant bioPROTAC platform capable of on-demand, targeted protein degradation using lipid nanocarriers. The final format, incorporating an N-terminal IpaH9.8 NEL and C-terminal ApP demonstrated robust, low nanomolar activity in 7 different cell lines and could be easily reprogrammed to polyubiquitinate diverse substrates including GFP, Ras, Erk, and Jnk. Strikingly, our bioPROTACs were active in various subcellular compartments including the cytosol, membrane, mitochondria, and nucleus. Finally, these biologic-based degraders were highly potent, exhibiting target elimination within hours of treatment. From a manufacturing standpoint, this format presents several key advantages. Firstly, our bioPROTACs are easily expressed using inexpensive bacterial cultures, enabling low- cost and accessible prototyping / testing of new degraders. Secondly, protein-based bioPROTACs exhibited no loss in activity for several weeks, are not susceptible to nuclease activity, and are amenable to typical storage conditions.

[0210] For Ras-targeting, it was found that the general trend for degradation rate was, in increasing order: siRNA < DNA < mRNA < protein. While this is in line with the expected result, it was noted that it is difficult to make direct comparisons of degradation rates between these modalities. For example, protein expression from plasmids is heterogeneous, and both DNA and mRNA can generate many protein molecules per nucleic acid obfuscating direct assessments against protein delivery. Moreover, siRNA-mediated degradation is dependent on the stability of their target protein and can have vastly different depletion rates. Nonetheless, rapid degradation was consistently observed using the herein described LNP-delivered bioPROTAC format and exhibited a target half-life of ~1.5 hours following treatment. Only small-molecule systems such as DTag, AID, or conventional PROTACs kinetics (<30 minutes), but these are burdened by many challenges as previously mentioned. To further highlight differences between modalities, an apparent hook effect was only observed with pcDNA bioPROTAC transfection. This is likely due to the inclusion of a strong CMV promoter and a 5’ Kozak sequence. These elements drive excessive bioPROTAC production to levels that are likely not reached by other delivery methods.

[0211] In addition to kinetic differences between degradation modalities, varying target specificity was also found depending on how the bioPROTAC was introduced. When transfected with mRNA encoding a Ras-targeting bioPROTAC, many non-target knockdowns were identified via MS / MS global proteome analysis. Comparatively, delivery of the same bioPROTAC as a recombinant protein induced fewer off-target effects. While some of the additional downregulated targets in theP-635623-PC mRNA group were identified as RNA-processing proteins, many others were not reliably explained. Taken together, the results herein support the idea that for some bioPROTACs, mRNA could induce more off-target degradation compared to protein delivery. Recently, pseudouridine- substituted mRNA was found to increase protein mistranslation, and if mutations in the DARPin variable region could generate off-target degraders from modified mRNA were investigated.

[0212] Although it has been successfully exploited as a C-terminal degradation domain, CHIP displayed no activity when fused to the N-terminus. This suggests that some E3 domains are more flexible than others for bioPROTAC development. Interestingly, it was also found that IpaH9.8- K27 was better than the previously-reported SPOP-K27 at eliminating Ras (Figure 3C, 3D). A similar result was recently published, showing that IpaH9.8 was more effective than SPOP at degrading GFP. In this work, this observation was confirmed, noting increased degradation rates for IpaH9.8 (1.5 hours) versus SPOP (>2.5 hours). In addition, it was found that SPOP-K27 could not be converted into an exogenously-delivered degrader (Figure 3G). It was hypothesized that this failure is due to a lower intrinsic potency and / or a reduced ability to complex with lipids caused by SPOP oligomerization. Thus, it was concluded that IpaH9.8 is a highly-active E3 domain which can be purified from bacterial cultures and remains active following lipid-mediated cytosolic delivery. The IpaH9.8 NEL is also tolerant to positioning within chimeric proteins, as it is now confirmed to be active in both N-terminal and C-terminal designs.

[0213] Towards extending bioPROTACs for clinical applications, polymeric nanogels have been harnessed for cytosolic antibody delivery and target degradation via TRIM-away. This method results in 50% degradation between 4-6 hours post-treatment. In another approach, the ZF5.3 CPP was appended to a BCL11A-targeting, SPOP-based degrader to enhance bioPROTAC internalization. With this system, 70% depletion of BCL11A was achieved within 12 hours using a final protein concentration in the micromolar range. In comparison to these two recent methods, the LNP / bioPROTAC-ApP approach described here is both faster and more potent, with the ability to reach >90% degradation within 5-6 hours using less than 100nM of protein. This efficiency was made possible through the combination of a highly-active IpaH9.8 module, negatively-charged ApP, and a compatible LNP vehicle.

[0214] From LNP screening results, it was noted that DOPE is a critical factor for bioPROTAC LNP performance, and it was observed improved LNP stability and increased degradation in formulations incorporating DOPE as a helper lipid. This is consistent with the DOPE’s known propensity to enhance endosomal escape by membrane fusion82. During therapeutic studies in MIAP-635623-PC PaCa-2 PDAC cells, it was noticed that some LNPs caused non-specific cytotoxicity at high doses which were alleviated by changing the ionizable lipid. This finding underscores the importance of tuning LNP properties based on cell line dependent responses. Crucially, future studies in animal models should carefully screen LNP formulations to simultaneously enhance target tissue uptake while reducing non-specific toxicity. Simultaneously, engineering of bioPROTACs for stronger degradation can lower the therapeutic dose, further reducing off-target effects.

[0215] In terms of cargo engineering, DARPins were highly-tolerant as chimeric fusion proteins, highlighting their promise for targeted degradation. In addition, degraders were able to be produced from various DARPin formats including N3C (3G124, K27) and N2C (J1 / 2_2_3, J1 / 2_2_25, EpE89) scaffolds, although a loop DARPin (012_F12) failed to degrade Bcl-xL.

[0216] Critically, it was found that IpaH9.8-fused bioPROTACs were subject to autoubiquitination leading to self-destruction upon cytosolic delivery. This effect did not restrict LNP:bioPROTAC efficacy in vitro, as potent degradation in cell assays was still observed. However, autoubiquitination could limit the ability of the herein described bioPROTAC to accumulate to therapeutic doses in vivo. One potential solution to relieve self-inhibition would be to identify lysine residues on the bioPROTAC (18 total on the Ras degrader) that are prone to self- targeting. These residues can then be selectively mutated to either alanine or arginine, rendering bioPROTACs resistant to self-degradation. Such an approach has demonstrated success in stabilizing other biodegraders.

[0217] Here, DARPins were used as model binders owing to their stability. Additionally, methods for screening diverse DARPins are well-established. Currently, the herein described system relies on published small protein sequences which have been selected for a handful of intracellular targets. However, this requirement is a major limitation for bioPROTAC design against targets for which no binder has been identified. Especially for cytosolic proteins, development of selective and high-affinity protein binders has not received the same level of attention compared to cell surface markers. If no existing binding protein exists for a target protein of interest, bioPROTAC development must be preceded by screening for target-specific binders using combinatorial scaffold libraries. Thus, it is expected that bioPROTAC development can serve as a compelling motivator for future selection of DARPins against intracellular targets. Other binding scaffolds including affibodies, nanobodies, and monobodies may also be tested in the herein described format for even broader versatility and application scope. With the ever-expanding collection of small protein scaffolds selected for antibody-like affinity and specificity, it is expectedP-635623-PC bioPROTACs will become more attractive as a therapeutic modality. The ability to deliver these protein-based degraders into cells serves as a promising avenue towards the treatment of many intractable diseases. Here, such a platform was developed, enabling cytosolic bioPROTAC protein delivery. While the results described herein demonstrate the feasibility of this modality, the technology is still in its early stages, and continued refinement of both the degrader and LNP carrier will be necessary for in vivo therapeutic applications.

[0218] Table 4 provides a list of DARPins that may be used in embodiments of the herein provided bioPROTACs. TABLE 4. DARPins that may be used in bioPROTACs according to embodiments of the present invention. DARPin Name Target Source 1800 p73 Münick, P., Zielinski, J., Strubel, A. et al. DARPins as a , n , n , nP-635623-PC 003_D9 BCL-W Johannes Schilling, Jendrik Schöppe, Andreas Plückthun, From DARPins to LoopDARPins: Novel LoopDARPin Desi n Allows the Selection of Low Picomolar Binders inCloning

[0219] All DNA fragments used in this study were synthesized as gBlocks (IDT; Coralville, IA), and codon optimization was performed using the IDT online codon optimization tool. For mammalian cell transfection experiments, gBlocks were codon-optimized for human cell line expression, and genes were inserted into a CMV-driven pcDNA3.1 vector with a 5’ Kozak sequence. Similarly, the lentivirus transfer plasmid was codon-optimized for human cell line expression. For bacterially-produced proteins, genes were codon-optimized for E. coli K12 expression and inserted into sortase tag-expressed protein ligation expression plasmid (pSTEPL). For cloning into pcDNA3.1 and pSTEPL, plasmids were linearized by double digestion with the appropriate restriction enzymes followed by backbone isolation using a QIAquick gel extraction kit (Qiagen; Germantown, MD). Plasmids for bacterial transformation and mammalian transfection were purified by Qiagen miniprep and Qiagen endotoxin-free maxiprep kits respectively. All plasmids were submitted for Sanger sequencing to confirm successful cloning of the correct protein sequence.

[0220] To generate the E3-deficient binder-only control, the anti-GFP DARPin, 3G124, was cloned into the linearized pcDNA3.1 backbone between KpnI and EcoRI using In-Fusion Snap Assembly master mix (Takara Bio; San Jose, CA). To produce the bioPROTAC mammalian- expression plasmids, an SPOP167–374-3G124 encoding gBlock was first inserted into the pcDNA3.1 backbone between KpnI and EcoRI by In-Fusion cloning. A GS-rich linker was included between SPOP and 3G124. In addition, a BamHI restriction site was designed into the gBlock between the two proteins for convenient cloning of alternate degradation domains. The remaining E3 sequences: SKP22-147, IpaH9.8254-545, SOCS2143-198, and CHIP128-303 were inserted into this plasmid between KpnI and BamHI by In-Fusion cloning. To generate Ras-targeting bioPROTAC mammalian expression vectors, gBlocks for SPOP167–374-DARPinK27 and SPOP167–374- DARPinK27n3 were first cloned into pcDNA3.1 between KpnI and EcoRI. Again, both GS-rich linkers and BamHI sequences were included between the E3 domain and DARPin domain, so thatP-635623-PC IpaH9.8254-545could be easily substituted for SPOP. The target plasmid: pcDNA3.1 GFP-KRAS was created by cloning KRAS into an eGFP-containing plasmid between BsrGI and EcoRV.

[0221] pSTEPL plasmids were previously produced for the expression of K27-D25-s11, K27- D30-s11, K27n3-D25-s11, and K27n3-D30-s11 in E. coli. The DARPin domains in these plasmids were replaced with 3G124 to produce 3G124-D25-s11 and 3G124-D30-s11. To generate SPOP- 3G124-D25-s11, a gBlock encoding SPOP167–374-3G124 was inserted into pSTEPL plasmids between NdeI and XhoI to retain the C-terminal ApP and s11 sequences. Using the resulting modular template, the remaining bioPROTAC expression plasmids were produced. Specifically, the IpaH9.8254-545 NEL sequence (wild-type or C337A mutant) was cloned between NdeI and BamHI, and the other target-specific DARPins (or null control): K27, K27n3, J1 / 2_2_3, J1 / 2_2_25, EpE89, and 012_F12 were cloned between BamHI and XhoI. To remove the D25 ApP sequence, pSTEPL plasmids were first digested with XhoI and AgeI. The backbones were then purified by gel extraction, and finally, a gBlock encoding the GFP s11 sequence was re-inserted at the same site. All proteins contained GS-rich linkers between E3, binding, ApP, and s11 reporter domains.

[0222] The GFP-KRAS lentiviral transfer plasmid was made by first linearizing the pLX304 vector (Addgene #25890) by PCR. Then, a gBlock encoding eGFP-KRAS was cloned into the pLX304 backbone using an In-Fusion HD cloning kit. The gene fragment contained a 5’ Kozak sequence and a GS-rich sequence between eGFP and KRAS proteins. To generate the iRFP-CaaX transfer plasmid, the iRFP sequence was amplified from DEST-H2B-iRFP670 (Addgene #90237) and cloned into a pHR lentiviral backbone upstream of a C-terminal CaaX sequence. Protein expression and purification

[0223] For all proteins, bacteria were cultured in 2YT autoinduction media including trace elements (Formedium; Norfolk, United Kingdom).

[0224] The following proteins were expressed in Shuffle T7 Express competent E. coli (C3029J, New England Biolabs; Ipswich, MA): SPOP-3G124-s11, SPOP-3G124-D25-s11, IpaH9.8- 3G124-D25-s11, SPOP-K27-D25-s11, SPOP-K27n3-D25-s11, IpaH9.8-K27-D25-s11, IpaH9.8- K27n3-D25-s11, IpaH9.8-J1 / 2_2_3-D25-s11, IpaH9.8-J1 / 2_2_25-D25-s11, IpaH9.8-EpE89- D25-s11, and IpaH9.8-012_F12-D25-s11. All other binders and bioPROTACs were expressed in T7 Express competent E. coli (C2566, New England Biolabs). Additionally, T7 Express E. coli previously transformed with pSTEPL eGFP was used in this study for recombinant GFPP-635623-PC expression. Expression cultures for SPOP-3G124-D25-s11, SPOP-K27-D25-s11, and SPOP- K27n3-D25-s11 were grown at 25°C for 48 hours. Expression cultures for IpaH9.8-J1 / 2_2_3-D25- s11, IpaH9.8-J1 / 2_2_25-D25-s11, IpaH9.8-EpE89-D25-s11, and IpaH9.8-012_F12-D25-s11 were grown at 30°C for 24 hours. All other proteins were expressed at 37°C for 24 hours. Cultures were grown in baffled flasks and shaken at 160-180 rpm. Using the STEPL bioconjugation / purification method, proteins were eluted from HisPur cobalt resin (Thermo Fisher Scientific) with triglycine (GGG). For some assays such as encapsulation or binding studies, dye- or biotin-labeled GGG peptides were used for C-terminal bioconjugation.

[0225] Following elution, proteins were further purified by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva; Marlborough, MA). Fractions were pooled and concentrated using Amicon Ultra centrifugal filters with a 10 kDa molecular weight cut-off (MilliporeSigma; Burlington, MA). The purity of the final product was characterized by SDS- PAGE, and protein concentration was determined using the BCA assay. Protein stocks were stored at -80°C for later use. Cell culture

[0226] The following cell lines: 293T, 293T GFP-KRAS, 293T GFP-KRAS / iRFP-CaaX, 293T GFP(1-10), A549, and HT1080 were either obtained from the inventors’ own stocks or produced for this study. HeLa cells stably expressing HaloTag-GFP-MITO, HaloTag-GFP-SPC25, HaloTag-GFP-MIS12, or HaloTag3x-GFP-TRF1 and U2OS cells stably expressing HaloTag3x- GFP-TRF1 were generously gifted by Michael Lampson. All of the above cell lines were cultured in DMEM (Thermo Fisher Scientific; Waltham, MA) supplemented with 1% pen-strep and 10% FBS. The colorectal cancer cell line, HCT116 was gifted by Michael Farwell and cultured in McCoy’s 5A medium (Thermo Fisher Scientific), and the pancreatic cancer line, MIA PaCa-2 was gifted by Gregory Beatty and cultured in RPMI (Thermo Fisher Scientific). Both McCoy’s 5A and RPMI media were supplemented with 1% pen-strep and 10% FBS. All cells were grown in a humidified incubator maintained at 37°C and 5% CO2 conditions. All native cell lines used in this study were authenticated by short tandem repeat (STR) profile analysis by the Penn Genomic and Sequencing Core. Stable cell line engineering

[0227] Lentivirus for GFP-KRAS transduction was produced using standard techniques. Briefly, equal amounts of pLX304 GFP-KRAS, pMD2.G (Addgene #12259), and psPAX2 (AddgeneP-635623-PC #12260) were transfected into 293T with Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. The virus-containing media was collected 48-hours later, centrifuged to remove cell debris, and passed through a 0.45µm PES syringe filter. Either 1mL, 0.5mL, or 100µL of clarified lentivirus supernatant was added to separate 293T cells seeded in 6- well plates in complete DMEM. After 24 hours, the culture media was exchanged for fresh DMEM. Once cells were confluent, GFP-positive populations were isolated with the assistance of the CHOP Flow Cytometry Core using a FACSJazz sorter (BD Biosciences; Franklin Lakes, NJ).

[0228] To generate 293T GFP-KRAS / iRFP-CaaX dual reporter cells, 293T GFP-KRAS cells were first plated overnight in a 6-well plate at 300,000 cells / well. The following day, culture media was replaced with complete DMEM containing 8µg / mL Polybrene, and 300µL of iRFP-CaaX lentivirus was added to cells and allowed to incubate overnight. Cells were grown until confluent and sorted with the assistance of the CHOP Flow Cytometry Core using a MoFlo Astrios cell sorter (Beckman Coulter; Brea, CA) for dual GFP / RFP positivity. mRNA synthesis

[0229] Production of mRNA was carried out by the Penn Institute for RNA Innovation mRNA Core. The coding sequence of IpaH9.8-DARPinK27 was codon-optimized using an in-house algorithm and cloned into an in vitro transcription (IVT) vector. Using IVT, mRNA was synthesized with a co-transcriptional 5' CleanCap and complete uridine-to-pseudouridine substitutions. LNP formulation

[0230] To formulate LNPs, an ethanol and an aqueous phase were mixed at a 1:3 volume ratio using a microfluidic device and pump 33 DS syringe pumps (Harvard Apparatus; Holliston, MA). To prepare the ethanol phase, ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phos-phocholine (DOPC), or 1,2-dioleoyl-sn-glycero-3- phosphoethanol-amine (DOPE), lipid-anchored polyethylene glycol (PEG) (Avanti Polar Lipids; Birmingham, AL), and cholesterol (Sigma; St. Louis, MO) components were combined. For protein encapsulating LNPs, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was included in the ethanol phase as a fifth component, and the aqueous phase was prepared using 1x PBS, shifted to pH 5 with 150 mM sodium chloride. For mRNA encapsulating LNPs, only the original four lipid components were included in the ethanol phase, and the aqueous phase was prepared using 10mM citrate buffer. After mixing, LNPs were subsequently dialyzed against 1x PBS for 1P-635623-PC hour to remove ethanol. LNP characterization

[0231] To determine hydrodynamic radius, LNPs were diluted 100x in 1x PBS in disposable cuvettes for dynamic light scattering (DLS) measurements on the Zetasizer Nano (Malvern Instruments; Malvern, UK). LNP size (Z-average diameter) and polydispersity index (PDI) are reported as the mean ± standard deviation of n = 3 measurements. To quantify surface zeta potential, LNPs were diluted 100x in water in DTA1070 zeta potential cuvettes (Malvern Panalytical, Malvern, UK) for measurement on the Zetasizer Nano instrument. mRNA concentration of mRNA encapsulating LNPs was determined using A260 / A280 absorbance measurements on a NanoQuant Plate (Tecan; Männedorf, Switzerland). For all LNP:protein formulations, the stated concentration refers to the total protein concentration (encapsulated and free) in solution based off the bioPROTAC input amount. Cryo-Electron Microscopy (cryo-EM) was performed by core personnel at the Beckman Center for Cryo-Electron Microscopy. LNP sample was concentrated 3-4x using Amicon Ultra Centrifugal Filters (10kDa MWCO, Millipore Sigma). Then, 3µL of concentrated LNPs were applied to a Quantifoil holey carbon grid which had been glow discharged. Grids were blotted, and plunge freezing was performed with liquid ethane using a Vitrobot Mark IV (Thermo Scientific). Imaging was performed on a Titan Krios (Thermo Scientific) equipped with a K3 Bioquantum. Protein encapsulation study

[0232] Proteins were labeled with a single C-terminal 5-carboxytetramethylrhodamine (5- TAMRA) dye via STEPL bioconjugation. Purified proteins were encapsulated into LNPs as described above and separated with columns packed with Sepharose CL-4B matrix (Cytiva). Fractions eluted by gravity chromatography were mixed with equal volumes of 0.1% Triton-X in black microwell plates and analyzed with a plate reader. Fractions corresponding to fluorescence peaks were pooled for DLS analysis. Plasmid DNA transfection

[0233] For E3 screening assays, 293T cells were plated at 100,000 cells / well in 24-well plates overnight. The following day, pcDNA plasmids for bioPROTAC and GFP-KRAS expression were separately diluted in Opti-MEM reduced serum medium (Thermo Fisher Scientific). In total, 6 dilutions of pcDNA bioPROTAC and 4 dilutions of pcDNA GFP-KRAS were made ranging from 0ng / well to 500ng / well. Dilutions of the bioPROTAC and target plasmids were mixed pairwise toP-635623-PC obtain 24 combinations of plasmids at 35µL final volume. Each of the 24 combinations was mixed with a separate solution comprising 2.5µL of Lipofectamine 2000 diluted into 35µL of Opti-MEM, and the resulting mixture was incubated at RT for 20 minutes to promote complexation. The DNA complexes were pipetted into wells, and plates was gently shaken to disperse the solution. After 8 hours, the culture media was replaced with fresh DMEM, and cells were grown for an additional 40 hours. At 48 hours post-transfection, cells were trypsinized and analyzed by flow cytometry.

[0234] For Western blot analysis and validation of Ras-targeting bioPROTACs, co-transfection was performed in a 6-well format with 600,000293T cells seeded per well. In these experiments, 0.5µg of target plasmid and 2µg of bioPROTAC plasmid (except IpaH9.8-based degraders) were co-transfected with 5-10µL of Lipofectamine 2000. The amount of IpaH9.8-3G124 varied for western blot analysis as indicated, and IpaH9.8-K27 / K27n3 was co-transfected at 0.25µg / well for flow cytometry. Cells were analyzed by western blotting or flow cytometry 24- or 48-hours post- transfection as indicated. Cytosolic protein delivery

[0235] In a typical protein delivery assay, cells were seeded overnight in either 6-well or 48- well plates such that they were 70-80% confluent at the time of transfection.

[0236] Lipofectamine-mediated delivery was performed in a 48-well format. First, stock proteins were diluted with Opti-MEM to achieve 20x the specified final treatment concentration in 10µL of Opti-MEM. Next, 2µL of Lipofectamine 2000 was diluted into 8µL of Opti-MEM, and the resulting mixture was thoroughly mixed with diluted protein by pipetting. The Lipofectamine / protein solution was allowed to complex for 15 minutes at RT, after which all 20µL of the mixture was added to wells containing 180µL of complete media. The corresponding lipid- free protein treatment samples were prepared by diluting stock proteins to 10x the indicated final concentration in 20µL of Opti-MEM.

[0237] LNP-mediated cytosolic protein delivery was performed in both 6-well and 48-well formats. In either format, LNP:protein was added directly to wells to achieve the desired final concentration. Flow cytometry

[0238] At specified time points following either protein delivery or nucleic acid transfection, cells were detached from plates with 0.25% trypsin and pelleted at RT in a table-top centrifuge at 500 g. Cell pellets were resuspended in FACS buffer (1x PBS, 1% w / v BSA, 1 mM EDTA) andP-635623-PC analyzed using a CytoFLEX flow cytometer (Beckman Coulter). At least 8000 cell-gated events were collected, and the geometric mean fluorescence intensity was calculated using FlowJo v10 software. For degradation assays involving 293T GFP-KRAS cells, data were normalized to untreated 293T GFP-KRAS cells and wild-type 293T cells. For split GFP delivery assays, the negative control sample was untreated GFP(1-10) cells, and the GFP-positive gate was defined such that only ~1% of the negative control sample would fall within that positive gate. Additionally, the geometric mean fluorescence intensity for each sample was divided by the mean fluorescence intensity of the negative control, and this ratio was taken as the fold-change MFI. siRNA-mediated knockdown

[0239] The day before transfection, 293T GFP-KRAS cells were plated overnight at 100,000 cells / well in a 24-well plate. Custom DsiRNA (IDT) designed to target KRAS were transfected into cells at a final concentration of 10nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific). An additional group was transfected with negative control DsiRNA (IDT) at a final concentration of 10nM. Transfections were performed according to manufacturer’s instructions. At indicated time points, cells were collected and analyzed by flow cytometry. The geometric mean fluorescence intensity was normalized to untreated and 293T wild-type samples, and the degradation rate of GFP-KRAS was estimated using a first-order decay model. The duplex sequences for siKRAS_1 are: 5’-rArCrGrArUrArCrArGrCrUrArArUrUrCrArGrArArUrCrATT- 3’ and 5’-rArArUrGrArUrUrCrUrGrArArUrUrArGrCrUrGrUrArUrCrGrUrCrA-3’. The duplex sequences for siKRAS_2 are: 5’-rGrGrArArUrUrCrCrUrUrUrUrArUrUrGrArArArCrArUrCAG- 3’ and 5’-rCrUrGrArUrGrUrUrUrCrArArUrArArArArGrGrArArUrUrCrCrArU-3’. mRNA delivery

[0240] LNPs encapsulating bioPROTAC mRNA were added directly into cell culture wells to achieve the indicated final concentration. Western blotting

[0241] Equal amounts of protein were boiled in sample loading buffer (928-40004, LI-COR Biosciences; Lincoln, NE) and resolved by SDS-PAGE with 4-12% Bolt Bis-Tris polyacrylamide gels (Thermo Fisher Scientific). Proteins were transferred onto PVDF membranes, blocked with Intercept TBS blocking buffer (LI-COR), and incubated with primary antibodies (diluted according to manufacturer’s recommendations) overnight at 4°C. The primary antibodies used in this study were: mouse anti-GFP (RT0265, Bio X Cell; Lebanon, NH), rabbit anti-Ras (3965, CellP-635623-PC Signaling Technology; Danvers, MA), rabbit anti-SAPK / JNK (9252, Cell Signaling Technology), rabbit anti-α-tubulin (2144, Cell Signaling Technology), rabbit anti-Erk1 / 2 (9102, Cell Signaling Technology), mouse anti-phospho-Erk1 / 2 (9106, Cell Signaling Technology), rabbit anti-Bcl-xL (2764, Cell Signaling Technology), and mouse anti-β-actin (3700, Cell Signaling Technology). Following primary antibody incubation, blots were incubated goat anti-rabbit 680RD (925-68071, LI-COR) and donkey anti-mouse 800CW (925-32212, LI-COR) IR-dye functionalized secondary antibodies (1:15,000 dilution). Membranes were scanned using an Odyssey M imaging system, and relative protein abundance was calculated by band densitometry using ImageJ software. Binding assays

[0242] To measure the binding affinity of modified DARPins, biotinylated proteins were first produced by STEPL using a GGG-biotin peptide. Proteins were further processed as described above, serially diluted, and incubated overnight in black, 96-well streptavidin coated plates at 4°C. The following day, 50µL of either GFP (A42613, Thermo Fisher Scientific) or KRAS (156968, Abcam; Boston, MA) diluted to 5µg / mL was added to wells, and plates were gently shaken for 1.5 hours at RT. Then, either rabbit anti-GFP (600-401-215L, Rockland Immunochemicals, 1:10,000 dilution; Limerick, PA) or rabbit anti-Ras (3339, Cell Signaling Technology, 1:1000 dilution) was incubated in wells for 1 hour at RT. Finally, wells were incubated with a 1:4000 dilution of goat anti-Rb Ab-HRP for 1 hour RT (31460, Thermo Fisher Scientific). Between each incubation step, wells were washed 3x with 200µL of wash buffer (1x PBS, 0.05% Tween 20), and all proteins were diluted in Superblock T20 buffer (Thermo Fisher Scientific). To detect HRP, the QuantaRed substrate kit (15159, Thermo Fisher Scientific) was used according to manufacturer’s instructions. Wells not receiving GFP / KRAS were used for background subtraction, and data were fit with a 4- parameter logistic model using GraphPad Prism v10. LDH cytotoxicity assay

[0243] The day before treatment, 293T cells were plated at 30,000 cells / well in a 96 well plate in 80µL of complete media. The following day, LNPs were serially diluted in PBS to 5x the indicated final treatment concentration, and 20µL of the diluted LNPs were added to media to achieve the specified final treatment concentration. Cells were kept in the incubator at 37°C for 8 hours before cytotoxicity was measured using the Lactate Dehydrogenase (LDH) assay kit (CK12, Dojindo Molecular Technologies; Gaithersburg, MD) according to manufacturer’s instructions. Cell viability was normalized to dead and live controls in GraphPad Prism v10.P-635623-PC Protein and LNP stability

[0244] For protein stability studies, purified bioPROTACs were stored at either -80°C or 4°C in PBS for at least 4 weeks prior to cytosolic delivery and degradation analysis. To assess LNP stability, LNP:bioPROTAC was stored at 4°C, and degradation efficiency was tested every 2 days. In vitro ubiquitination assay

[0245] For each 25µL reaction, a master mix was made by combining 2.5µL of 10x reaction buffer (500mM HEPES, 500mM NaCl, 10mM TCEP), 1µL of ubiquitin (U-100H, R&D Systems; Minneapolis, MN), 2.5µL of MgATP solution (B-20, R&D Systems), 0.5µL of the human E1, UBE1 (E-304, R&D Systems), 1µL of the human E2, UbcH5b / UBE2D2 (E2-622, R&D Systems), 3µL of human KRAS (156968, Abcam), and 13.25µL of MQ H2O. To this master mix, 1.25µL of either 10µM bioPROTAC or 10µM control bioPROTAC was added and mixed well, initiating polyubiquitination. An additional negative control was included where 1.25µL of MQ H2O was added to the master mix instead of bioPROTAC. Reaction mixtures were incubated for 2 hours at 37°C and quenched with 25µL of 2x Tricine-SDS buffer. To detect ubiquitinated KRAS, at least 0.5µL of sample (15ng KRAS) was resolved by SDS-PAGE and probed by western blotting with an anti-KRAS antibody. To assay bioPROTAC autoubiquitination, proteins were first labeled with a single C-terminal TAMRA dye via STEPL, and in vitro ubiquitination was performed in the absence of target protein. Samples were resolved by SDS-PAGE, and ubiquitin-modified bioPROTACs were visualized by fluorescence imaging. Sample preparation for proteomic analysis

[0246] Cells were harvested by scraping, pelleted by centrifugation, washed twice with ice-cold PBS, and snap frozen. Proteins were extracted by adding 200µL of ice-cold lysis buffer (8M urea, 75mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, protease inhibitor cocktail) to the cell pellet followed by 3 cycles of incubation on ice (5 minutes) and vortexing (10 seconds). Finally, samples were sonicated with an ultrasonic homogenizer and clarified by centrifugation at 20,000 g for 10 minutes, and the supernatant was collected for further analysis. Protein concentration was measured using a BCA Protein Assay Kit (A53227, Thermo Fisher Scientific), and equal amounts of protein (200µg) were taken from each sample. Samples were reduced using 5mM dithiothreitol (DTT) at 60°C for 30 minutes followed by cysteine alkylation using 20mM iodoacetamide (IAA) for 15 minutes in the dark. Samples were diluted to 1M Urea using 50mM Tris-HCl, pH 8.0, andP-635623-PC trypsin digestion was performed with modified sequencing-grade trypsin (Promega, Madison, WI) at a 1:30 trypsin-to-protein ratio overnight at 37°C. Basic reverse phase liquid chromatographic (bRPLC) fractionation of digested peptides and C18 clean-up

[0247] Peptide fractionation was carried out using a C-18 StageTip protocol eluting with a high pH mobile phase (50mM Triethylammonium bicarbonate pH 8.5 (TEABC)). Briefly, the C-18 material was packed into 200μl pipette tips, activated with 100% acetonitrile (ACN), and equilibrated with 50mM TEABC. Peptides were resuspended in 50mM TEABC and loaded onto the C-18 StageTips column. Samples were passed twice through the column, followed by washing with 50mM TEABC. Finally, samples were eluted with increasing concentrations of ACN (5-50%) in TEABC to yield a total of 8 fractions and combined pairwise (1+5, 2+6, 3+7, 4+8) to give a total of 4 fractions. Peptide desalting was carried out with the C-18 StageTip method. The C-18 material was packed into 200μl pipette tips, activated with 100% ACN, and equilibrated with 0.1% formic acid in ACN. Fractionated peptide samples were resuspended in 0.1% formic acid and loaded onto the C-18 StageTip. Samples were passed through the column twice, followed by washing with 0.1% aqueous formic acid in ACN and elution with 40% ACN in aqueous 0.1% formic acid. The eluent was dried and stored at −20°C until LC-MS / MS analysis. Liquid chromatography tandem mass spectrometry (LC-MS / MS)

[0248] Cleaned peptides were analyzed on Thermo Scientific Orbitrap Exploris 240 mass spectrometer interfaced with Thermo Scientific UltiMate 3000 HPLC and UHPLC Systems. Peptide digests were reconstituted in 0.1% formic acid and were separated on an analytical column (75 µm × 15 cm) at a flow rate of 300nL / min using an increasing gradient of solvent B (0.1% formic acid in 100% acetonitrile). The total run time was set to 120 min. The mass spectrometer was operated in data-dependent acquisition mode. A survey full scan MS (m / z 400–1600) was acquired in the Orbitrap with a resolution of 6000 normalized AGC target of 300%. Data were acquired in topN with 20 dependent scans. Ions were fragmented using 37% normalized collision energy and detected at a mass resolution of 1500. Dynamic exclusion was set for 8s with a 10ppm mass window. Proteomic data analysis

[0249] MS / MS searches were performed with SEQUEST against the Uniprot database for human proteins supplemented with bioPROTAC sequences using Proteome Discoverer (Version 3.0,P-635623-PC Thermo Fisher Scientific; Bremen, Germany). The workflow included Spectrum files, Spectrum selector, SEQUEST search nodes, target decoy PSM validator, peptide validator, event detector, precursor quantifier. Data was searched in label free quantification mode using unique peptides for quantification. Oxidation of methionine and N-terminal protein acetylation were used as dynamic modifications and carbamidomethylation of cysteine was set as a static modification. MS and MS / MS mass tolerances were set to 10 ppm and 0.05 Da, respectively. A maximum of two missed cleavage was allowed. Target-decoy database searches used for calculation of false discovery rate (FDR) and for peptide identification FDR were set at 1%. Feature mapper and precursor ion quantifier were used for label-free quantification. Custom R scripts were used for downstream data analyses and visualizations. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE85partner repository with the dataset identifier PXD049388. Size exclusion chromatography for protein interaction analysis

[0250] Recombinant eGFP and binding partners were diluted with PBS to a final concentration of 10µM per protein in a final volume of 200µL. Samples were incubated at RT for 10 minutes and centrifuged at 12,000 g to remove aggregates. Protein mixtures were injected into an ӒKTA pure chromatography system (Cytiva) with a Superdex 200 Increase 10 / 300 GL column installed. Eluted fractions were pooled and analyzed by SDS-PAGE. Fluorescence microscopy of HeLa and U2OS

[0251] HeLa and U2OS cells expressing GFP-fusion proteins were either left untreated or treated with 100nM K1:bioPROTAC targeting GFP. After 7.5 hours, cells were stained with Hoechst 33342 at 1µg / mL for 20 minutes. After nuclei staining, media was aspirated and replaced with FluoroBrite DMEM (A1896701, Thermo Fisher Scientific). Cells were imaged using a Nikon Ti2- E microscope equipped with a Yokagawa CSU-W2 spinning disk, and an sCMOS camera (Photometrics). Images were acquired using a 20x air objective and the 405nm and 488nm laser lines for Hoechst and GFP respectively. Images were background subtracted and equalized with ImageJ software. Time-course confocal microscopy

[0252] 293T GFP-KRAS / iRFP-CaaX cells were seeded overnight at 100,000 cells / well in glass 96-well plates treated with 10µg / mL human plasma fibronectin (FC010, Thermo Fisher Scientific). The following day, LNPs were pipetted directly into wells, and cells were imaged every 20 minutes for 12 hours using a 40x air objective. Throughout the duration of the experiment,P-635623-PC temperature and CO2were maintained at 37°C and 5% respectively using an environmental chamber (Okolab; Sewickley, PA). Images were acquired using a Nikon Ti2-E microscope equipped with a Yokagawa CSU-W1 spinning disk, 405 / 488 / 561 / 640 nm laser lines, and an ORCA-fusionBT digital camera C15440 (Hamamatsu). Time-course image analysis

[0253] Image pre-processing was performed using ImageJ with the MorphoLibJ plugin (https: / / imagej.net / plugins / morpholibj). First, GFP and RFP channel images were equalized, and GFP channel images were further background subtracted using the rolling-ball algorithm. To segment individual cells, a morphological closing operation (octagon element, radius = 15 pixels) was applied to RFP channel images, and a watershed algorithm was implemented (tolerance = 200). The resulting catchment basins were extracted and loaded into CellProfiler87as cell objects, while processed GFP-channel images were uploaded as grayscale images. Finally, a custom pipeline was used to measure the integrated and membrane-associated fluorescence intensity of individual cells. Between 400-500 single cells were analyzed at each time point and their calculated fluorescence intensities were averaged and normalized to fluorescence intensity at t = 0. Real-time cell viability assay

[0254] Cells were plated in a 96-well E-plate (Agilent; Santa Clara, CA) and allowed to attach overnight. The following day LNPs were added to wells to achieve the indicated final protein or mRNA concentrations, and proliferation was monitored using the xCELLigence Real-Time Cell Analysis system (Agilent). The cell index, an arbitrary unit of confluence, was normalized to untreated controls for analysis. Protein structural model

[0255] The structure of the Ras-targeting IpaH9.8-K27 bioPROTAC was predicted by AlphaFold88. The resulting model was then aligned with IpaH9.8 NEL (PDB ID: 6LOL) and DARPinK27 (PDB ID: 5O2S) with PyMol. Statistics

[0256] Multiple batches of proteins and LNPs were used throughout this study. Two-tailed t tests and ANOVA were used for data analysis. When appropriate, multiple-comparisons testing was performed, and Bonferroni correction was applied. All statistical analyses were performed using GraphPad Prism v10.

Claims

P-635623-PC What is claimed is:

1. A recombinant fusion protein comprising an E3 ubiquitin ligase domain, a small protein binding scaffold, and an anionic polypeptide, wherein the small protein binding scaffold targets an intracellular protein that is targeted to be degraded intracellularly.

2. The fusion protein of claim 1, wherein the E3 ligase domain comprises a mammalian E3 ligase, an E3 ligase adapter, or bacterial novel E3 ligase.

3. The fusion protein of claim 2, wherein the mammalian E3 ligase is a Really Interesting New Gene (RING) E3 ligase, Homologous to E6AP C-terminus (HECT) E3 ligase, a RING- between-RING (RBR) E3 ligase, or an U-box E3 ligase.

4. The fusion protein of claim 2, wherein the E3 ligase adapter is SPOP, VHL, SOCS2 or SKP2.

5. The fusion protein of claim 3, wherein the RING E3 ligase is COP1, Mdm2, or TRAF6.

6. The fusion protein of claim 3, wherein the HECT E3 ligase is E6AP, HUWEI, or HERC.

7. The fusion protein of claim 3, wherein the RBR E3 ligase is HHARI or UBAC.

8. The fusion protein of claim 3, wherein the U-box E3 ligase is CHIP.

9. The fusion protein of claim 2, wherein the bacterial novel E3 ligase is SlrP, SspH1, SspH2, SspH3, IpaH1.4, IpaH 2.5, IpaH 4.5, IpaH 7.8, IpaH 9.8, IpaH a, IpaH b, IpaH c, IpaH d, IpaH e, NopM, RipAR, RipAW, RipV1, or RipV2.

10. The fusion protein of claim 1, wherein the small protein binding scaffold is a designed Ankyrin repeat protein (DARPin), a nanobody, an affibody, a monobody, a nanobody, a knottin, an affimer, a Nanofitin, an avimer, or a Centyrins.

11. The fusion protein of claim 10, wherein the DARPin is DARPin 3G124, DARPin K27, DARPin K19, DARPin K13, DARPin EpE89, DARPin J1 / 2_2_3, or DARPin J1 / 2_2_25.

12. The fusion protein of claim 1, wherein the anionic polypeptide comprises at least 50% negatively charged amino acids.

13. The fusion protein of claim 1, wherein the anionic polypeptide comprises aspartic acid, glutamic acid, or a combination thereof.

14. The fusion protein of claim 13, wherein the anionic polypeptide comprises about 10-50P-635623-PC aspartic acid, about 10-50 glutamic acid, or about 10-50 amino acids comprising aspartic acid and glutamic acid.

15. The fusion protein of claim 1, wherein the intracellular protein is a Ras protein, a MAPK protein, a p53 protein, a Myc protein, a NF-κB protein, β-catenin, a Tau protein, an amyloid beta (Aβ) protein, estrogen receptor, androgen receptor, multidrug resistance proteins (MRP), B-cell lymphoma-2 (BCL2) family, Cyclin D1 protein, AKT protein, Mdm2 protein, Surviving protein, E2F, HIF-1alpha, STAT family, TNF, interleukin family, NPPB, NPPA, C-reactive protein, TGFbeta, alpha-synuclein, a p73 protein, and / or mucin.

16. The fusion protein of claim 15, wherein (a) the Ras protein selected from the group consisting of KRAS, HRAS and NRAS; (b) the MAPK protein is selected from the group consisting of Erk and pErk; (c) the Myc protein selected from the group consisting of c-myc (MYC), I-myc (MYCL) and n-myc (MYCN); and (d) the NF-κB protein selected from the group consisting of NF- κB1, NF- κB2, relA, RelB and c-Rel.

17. The fusion protein of claim 1, wherein the E3 ligase domain is operably linked to a N- terminus of the small protein binding scaffold, or the E3 ligase domain is operably linked to a C- terminus of the small protein binding scaffold.

18. The fusion protein of claim 1, wherein the anionic polypeptide is operably linked to a C- terminus of the small protein binding scaffold, or the anionic polypeptide is operably linked to a N-terminus of the small protein binding scaffold.

19. A composition comprising the fusion protein of claim 1 and a cationic agent that facilitates cytoplasmic delivery of said fusion protein.

20. The composition of claim 19, wherein the cationic agent is a nano-carrier or an ionizable carrier, wherein said nano-carrier or ionizable carrier comprises ionizable-lipid, cationic lipid, polymer, or combination thereof.

21. The composition of claim 20, wherein the nano-carrier is an ionizable lipid-like nanoparticle (LNP).P-635623-PC 22. The composition of claim 21, wherein the LNP comprises about 30-50% ionizable or cationic lipid, about 30-50% cholesterol, about 10-15% neutral lipid, and about 1-5% PEG-lipid, wherein the percentages are molar percentages.

23. A method of targeted intracellular degradation of a target protein, comprising contacting a cell with a composition comprising the fusion protein of claim 1 and a cationic agent that facilitates cytoplasmic delivery of said fusion protein, wherein the small protein binding scaffold of said fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of said fusion protein.

24. A method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a composition comprising the fusion protein of claim 1 and a cationic agent that facilitates cytoplasmic delivery of said fusion protein, wherein the small protein binding scaffold of said fusion protein targets an intracellular protein that is targeted to be degraded intracellularly by a process mediated by the E3 ubiquitin ligase domain of said fusion protein, thereby treating said disease or condition in said subject.

25. The method of claim 24, wherein the disease or condition is cancer, sepsis, inflammatory disease, or neurodegenerative disorder.

26. The method of claim 25, wherein the cancer is a Ras-driven cancer.

27. The method of claim 25, wherein the cancer is pancreatic cancer, non-small cell lung cancer, or colorectal cancer.

28. The method of claim 25, wherein the inflammatory disease is arthritis.

29. The method of claim 25, wherein the neurodegenerative disorder comprises neurofibril degradation.

30. The method of claim 26, wherein intracellular protein degradation is proteasomal and / or lysosomal degradation.

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