Liver support systems and methods of use

Abstract

The present disclosure relates to liver support systems comprising (a) an apheresis column; and (b) a detoxification column comprising a plurality of liver tissues, wherein the liver support system is configured to allow a fluid to flow from the apheresis column to the detoxification column. The detoxification column can provide in detoxification, secretory and regenerative function. Detoxification can include promoting the central urea cycle which is severely compromised and CYP function which is critical for detoxification of xenobiotics, cellular metabolism and homeostasis. The apheresis column can function to adsorb granulocytes from the fluid and to protect the plurality of liver tissues from inflammatory damage. The present disclosure relates to methods of using the liver support systems and engineered cells for use in the liver support systems.

Description

Attorney Docket No. KNZ-001WO LIVER SUPPORT SYSTEMS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 507,225, filed on June 9, 2023, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] Acute-on-chronic liver failure (ACLF) is characterized by devastating clinical symptoms, with a 28-day mortality rate of 20% for ACLF-1, 40-60% for ACLF-2, and 80% for ACLF-3. ACLF represents acute liver failure (ALF) / organ failure in the setting of progressive chronic liver disease, accompanied by systemic inflammation and progressive multi-organ dysfunction. In the US, there are approximately 3 million patients living with cirrhosis, among whom approximately 500,000 have evidence of decompensation (i.e., clinical complications). Each year, approximately 50,000 patients progress to ACLF. The most common causes include viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), and other immune-mediated liver diseases. However, in recent years, the incidence of NASH has been increasing globally, leading to a significant predicted increase in ACLF incidence over the next 20 years. ALF occurs in the absence of chronic liver disease and is usually associated with overdose of acetaminophen or other hepatotoxic medications. Mortality rates in ALF are high when the degree of liver damage is severe despite best supportive care. Therefore, there is a significant unmet need for new therapies that can reduce the mortality rate caused by ACLF and ALF, decrease the duration of intensive care unit (ICU) management, and promote liver recovery. The goal of this development program is to provide liver function support using innovative cell therapy / device products, aiming to either ensure disease self-recovery or serve as a bridge to liver transplantation in order to reduce mortality rates.

[0003] The only currently known curative treatment for ACLF patients is liver transplantation, however, in the US, only about 8000 liver transplantations are performed annually. The current standard treatment for ACLF patients includes supportive ICU management (for ACLF-2 and -3) and treatment of underlying causes and precipitating factors (such as antimicrobial therapy for sepsis and medical management of hepatic 1 IPTS / 128597941.1Attorney Docket No. KNZ-001WO encephalopathy and variceal bleeding). Specific treatment options include the use of terlipressin for ACLF-1 / hepatorenal syndrome (with recovery of renal function observed in 30% of patients) and interventions such as plasma exchange and hemofiltration for improving multi-organ dysfunction associated with ACLF-2 and -3.

[0004] Therefore, there is a need for new treatment strategies that can reduce mortality, shorten ICU management periods, and promote liver recovery for ACLF / ALF patients, as the current therapeutic options, including liver transplantation, have limitations and are not readily available to all patients. The major reasons for failure of prior efforts of extracorporeal liver support systems including bioartificial liver devices include poor viability of cells that provide adequate detoxification, synthetic and regenerative function, and inability to address systemic inflammation. SUMMARY

[0005] Disclosed herein, in various embodiments is, a liver support system comprising, in sequence: (a) an apheresis column; and (b) a detoxification column comprising a plurality of liver cells or tissues, wherein the liver support system is configured to allow a fluid to flow from the apheresis column to the detoxification column.

[0006] In some embodiments, the apheresis column is configured to adsorb one or both of granulocytes and monocytes from the fluid. In some embodiments, the apheresis column is a granulocyte and monocyte adsorption apheresis (GMA) column. In some embodiments, the GMA column comprises cellulose acetate. In some embodiments, the apheresis column reduces toxicity of the liver cells or tissues. In some embodiments, the apheresis column comprises one or more modifications for enhanced leukoadsorption.

[0007] In some embodiments, the detoxification column is configured to remove toxic substances from the fluid, provide factors that maintain liver function, and / or to promote regeneration of a damaged liver. In some embodiments, the fluid is whole blood. In some embodiments, wherein the blood is human blood. In some embodiments, the fluid is plasma.

[0008] In some embodiments, the device further comprises a plasma separator between the apheresis column and the detoxification column.

[0009] In some embodiments, the liver tissues comprise cells selected from the group consisting of: pluripotent stem cell-derived induced hepatocyte-like cells aggregates or organoids (iHLCs), liver bud organoids, liver organoids, primary hepatocytes, primary liver 2 IPTS / 128597941.1Attorney Docket No. KNZ-001WO organoids, primary ductal organoids, fetal hepatocytes, hepatic progenitors, hepatoblasts, stem cell-derived hepatocytes, and directly reprogrammed hepatocytes (hiHep).

[0010] In some embodiments, the liver tissues comprise iHLCs. In some embodiments, the iHLCs are engineered human cells, optionally induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the iHLCs comprise one or more genetic alterations to confer resistance to oxidative stress.

[0011] In some embodiments, the one or more genetic alterations to confer resistance to oxidative stress comprise expression of a constitutively active variant of nuclear factor erythroid 2-related factor 2 (NRF2), mouse L-gluconolactone oxidase (mGULO), mutant albumin domain 2 comprising F211R and R218L amino acid substitutions, or MSRP5. In some embodiments, the iHLCs comprise one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling. In some embodiments, the one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling comprise: (a) edits to MHC class-I and / or MHC class-II genes; (b) suppression of expression of HLA-A, HLA-B, B2M, NLRP3, and / or CIITA; and / or (c) induction of expression of HLA-E and / or anti-IL1b. In some embodiments, the one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling comprise reduced or substantially no expression of one or more protein selected from the group consisting of HLA-A, HLA-B, and CIITA. In some embodiments, the iHLC has reduced or substantially no expression of each of HLA-A, HLA-B, and CIITA. In some embodiments, the expression of the one or more proteins is reduced by gene editing. In some embodiments, the expression of the one or more proteins is reduced by CRISPR-Cas9-mediated gene editing. In some embodiments, the iHLCs comprise one or more genetic alterations to improve longevity of the iHLCs. In some embodiments, the one or more genetic alterations to improve longevity comprise one or more alterations to genes involved in cell cycle, apoptosis, and / or metabolism. In some embodiments, the one or more alterations to genes involved in metabolism comprise alterations to genes involved in fatty acid oxidation. In some embodiments, the iHLCs comprise one or more genetic alterations to improve detoxification function and / or hepatic synthetic function of the iHLCs. In some embodiments, the one or more genetic alterations to improve detoxification function and / or hepatic synthetic function comprise: (a) one or more genetic alterations to genes involved in albumin affinity, albumin functionality, urea cycle, and / or bilirubin clearance; coagulation factor genes and / or cytochrome P450 genes; and / or (b) suppression of expression of ACMSD, CD38, PARP, and / or CD73. In some 3 IPTS / 128597941.1Attorney Docket No. KNZ-001WO embodiments, the iHLCs comprise one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver. In some embodiments, the one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver comprise ectopic expression of HNF4A, TBX3, FOXA1, FOXA2, FOXA3, FOXA1, PROX1, HNF1A, HNF1B, CEBPA, NR1I2, KDM2B, GATA4, HNF6, ATF5, HHEX, NANOG, SOX2, OCT3, OCT4, c-and / or c-MYC, CHIR99021, RepSox, Forskolin, Valproic acid, Parnate, TTNPB, and / or DZNep.

[0012] In some embodiments, the iHLCs comprise inducible expression of one or more anti-apoptotic proteins. In some embodiments, the anti-apoptotic protein is a BCL2 family protein. In some embodiments, the anti-apoptotic protein is selected from the group consisting of BCL2, BLC2L1 (BCL-XL), BAX, IAP1, BIRC3,XIAP, NAIP, BIRC5, BIRC7, ILP2, and BIRC6. In some embodiments, the anti-apoptotic protein is BCL2. In some embodiments, the anti-apoptotic protein is BLC2L1 (BCL-XL).

[0013] In some embodiments, the iHLCs are comprised within a two-dimensional culture, a three-dimensional culture, a cellular aggregate, a tissue section, or one or more liver organoids. In some embodiments, the iHLCs are comprised within one or more liver organoids. In some embodiments, the iHLCs are encapsulated within alginate capsules. In some embodiments, the system comprises one or more modifications to reduce mast cell activity. In some embodiments, the apheresis column comprises one or more modifications for enhanced leukoadsorption.

[0014] In some embodiments, the device further comprises a plasma separator between the apheresis column and the detoxification column. In some embodiments, the system is configured for the fluid to flow at a rate of 30-600 mL / hr. In some embodiments, the system comprises a peristaltic pump. In some embodiments, the system is configured to allow to fluid to flow (1) from a subject, (2) to the apheresis column, (3) to the detoxification column, and (4) back to the subject.

[0015] Also disclosed herein, in various embodiments, is a method of treating a liver disease in a subject in need thereof, the method comprising flowing whole blood from the subject through the liver support system disclosed herein. In some embodiments, the subject is afflicted with is acute-on-chronic liver failure (ACLF), acute liver failure (ALF), liver failure due to genetic inborn errors of metabolism, liver failure due to mitochondrial disease, acute fatty liver of pregnancy, intractable pruritus., drug-induced liver injury (DILI), ischemic hepatitis, cholestatic liver disease, or liver failure or liver injury following liver 4 IPTS / 128597941.1Attorney Docket No. KNZ-001WO mass resection. In some embodiments, the liver disease is ACLF. In some embodiments, the ACLF is ACLF-1, ACLF-2, or ACLF-3. In some embodiments, the subject is afflicted with viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis of any etiology, sepsis, or systemic inflammatory response syndrome. In some embodiments, the subject is awaiting liver transplant. In some embodiments, the subject has undergone liver transplant or liver resection. In some embodiments, the subject is receiving or has received terlipressin, continuous renal replacement therapy, plasmapheresis, blood filtration dialysis, and / or plasma exchange. In some embodiments, the subject’s blood is flown through the liver support system for a period of at least two hours, at least three hours, at least six hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 38 hours, at least 42 hours, or at least 48 hours.

[0016] Also disclosed herein, in various embodiments, is an engineered hepatocyte-like cell comprising: (a) reduced or substantially no expression of one or more protein selected from the group consisting of HLA-A, HLA-B, and CIITA; and (b) engineered, optionally inducible, expression of one or more anti-apoptotic proteins. In some embodiments, the anti- apoptotic protein is a BCL2 family protein. In some embodiments, the anti-apoptotic protein is selected from the group consisting of BCL2, BLC2L1 (BCL-XL), BAX, IAP1, BIRC3,XIAP, NAIP, BIRC5, BIRC7, ILP2, and BIRC6. In some embodiments, the anti- apoptotic protein is BCL2. In some embodiments, the anti-apoptotic protein is BLC2L1 (BCL-XL). In some embodiments, expression of the one or more anti-apoptotic protein is under the control of a doxycycline-inducible promoter. In some embodiments, the cell has reduced or substantially no expression of each of HLA-A, HLA-B, and CIITA. In some embodiments, wherein the expression of the one or more proteins is reduced by gene editing. In some embodiments, wherein the expression of the one or more proteins is reduced by CRISPR-Cas9-mediated gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG.1 depicts a schematic of a liver support system disclosed herein, according to an embodiment. The liver support system include an apheresis column (red, top) and a detoxification column (gold, bottom). The detoxification column can provide one or more of detoxification, secretory, and regenerative function.

[0018] FIG.2A depicts a schematic diagram of the generation of iHLC derived from HLA-triple knockout (TKO) human iPSCs. Posterior gut endoderm cells were induced using 5 IPTS / 128597941.1Attorney Docket No. KNZ-001WO HLA-TKO iPSCs, which can be cryopreserved and expanded (reference). FIG.2B depicts representative images of iHLC derived from HLA-A / B wild-type and CIITA wild-type human iPSC lines (1383D6 and Ff-XT28s05_cont) and HLA-A / B knockout and CIITA knockout lines (Ff-XT28s05-Abo_To #14-4 and Ff-I01s04-AbII-KO-54). Bar, 50 mm. WT, wild-type; KO, knockout. FIG.2C depicts flow cytometry analysis of iHLC derived either from HLA-A / B, CIITA WT or KO human iPSC lines, which were stained with HLA-A24 and HLA-B7 antibodies after IFN-g treatment.

[0019] FIG.3A depicts a schematic diagram of the generation of encapsulated iHLC derived from HLA-TKO human iPSCs. FIG.3B depicts images of encapsulated iHLC determined stained with calcein-AM (live cells) / EthD-1 (dead cells). The organoids cultured in Matrigel were set as a control. FIG.3C depicts a graph showing albumin secretion from iHLCs cultured in Matrigel and encapsulated in alginate, which was measured using the culture supernatant for 24 hr in vitro.

[0020] FIG.4 depicts an assay measuring the viability of encapsulated HepG2 spheroids housed in a column following extracorporeal circulation of a rat.

[0021] FIG.5A depicts images of encapsulated iHLC housed in the column of a liver support system following extracorporeal circulation of an ACLF rat model stained for cleaved caspase-3 and F-actin. Encapsulated iHLC without extracorporeal circulation was used as a control. FIG.5B depicts a graph showing quantification of the percentage of cleaved caspase-3-positive apoptotic iHLCs after extracorporeal circulation in the ACLF rat model shown in (H). Statistical significance between iHLC column only and iHLC+GMA columns conditions (*P=0.0198), one-way ANOVA followed by Tukey’s post hoc test. FIG. 5C depicts a graph showing quantification of cell-free human mitochondrial DNA (mtDNA) in plasma of ACLF model rats that received the extracorporeal circulation treatment. Data are shown as average ± SD. Statistical significance between control and iHLC only conditions (*P=0.0010), and between iHLC only and iHLC+GMA conditions (*P=0.0475), one-way ANOVA followed by Kruskal-Wallis test.

[0022] FIG.6A depicts a schematic diagram depicting the configuration of the HLA- TKO iHLC-populated liver support system, where the GMA and encapsulated HLA-TKO iHLC columns were connected in tandem, to ACLF model rat (upper panel). One hour after intraperitoneal lipopolysaccharide (LPS) injection into rats with bile duct ligation (BDL), the treatment of extracorporeal circulation was performed for 2 hours (lower panel). FIG.6B depicts a Kaplan-Meier survival curve of BDL+LPS rats treated with no therapy, empty 6 IPTS / 128597941.1Attorney Docket No. KNZ-001WO column, GMA column only, iHLC column only, and tandemly connected GMA-iHLC columns. *P=0.0093, log-rank (Mantel-Cox) test. FIG.6C depicts graphs showing relative changes in plasma levels of ammonia (NH3), AST, ALT, and total bilirubin (T-bil) at 5 hours post the liver support system treatment compared to those at 0 hours. Average ± SD, *P<0.05, Student’s t test. FIG.6D depicts images of empty column- liver support system and GMA-iHLC -liver support system -treated BDL+LPS rats at 24 hours after the treatment.

[0023] FIG.7A depicts representative images of H&E staining and Ki67 immunostaining of liver histology of BDL+LPS rats in empty column-liver support system and GMA-iHLC -liver support system treated groups (72 hours post extracorporeal circulation treatment). Bar, 100 mm. FIG.7B depicts quantification of Ki67 staining of the liver in empty column-liver support system and GMA-iHLC -liver support system treated groups. Average ± SD, *P=0.0026, Student’s t test. FIG.7C depicts representative images of p21 immunostaining of liver histology of BDL+LPS rats in empty column-liver support system and GMA-iHLC -liver support system treated groups (24 hours post extracorporeal circulation treatment). Bar, 100 mm.

[0024] FIG.8A depicts a volcano plot of differentially expressed genes (DEGs) in empty column-liver support system and GMA-iHLC -liver support system treated BDL+LPS rat livers analyzed by RNA-seq. FIG.8B depicts gene set enrichment analysis of significantly upregulated genes in GMA-iHLC -liver support system group compared to empty column- liver support system group. Descartes Cell Types and Tissue 2021 was used as a reference gene set, showing enrichment of the upregulated genes related to hepatoblasts in liver. FIG. 8C depicts enrichment analysis of ENCODE and ChEA consensus transcription factor and transcription factor perturbation gene sets for upregulated genes by GMA-iHLC -liver support system treatment.

[0025] FIG.9A depicts representative images of H&E staining and MPO immunostaining of liver histology of BDL+LPS rats in empty column-liver support system and GMA-iHLC -liver support system treated groups (24 hours post extracorporeal circulation treatment). Bar, 100 mm. FIG.9B depicts a graph of liver injury scores of the liver tissues in BDL+LPS rats treated with empty column-liver support system and GMA- iHLC -liver support system 24 and 72 hours post-treatment. FIG.9C depicts representative images of TUNEL staining and cleaved caspase-3 immunostaining of liver histology of BDL+LPS rats in empty column-liver support system and GMA-iHLC -liver support system treated groups (72 hours post extracorporeal circulation treatment). Bar, 100 mm. 7 IPTS / 128597941.1Attorney Docket No. KNZ-001WO

[0026] FIG.10A depicts a Venn diagram showing the number of up / down-regulated genes in empty column-liver support system BDL+LPS rat liver vs. normal liver and up / down-regulated genes in GMA-iHLC -liver support system vs. empty column-liver support system BDL+LPS rat liver DEGs. The overlapping region indicated the numbers of expressed genes reverted by GMA-iHLC -liver support system treatment. FIG.10B depicts gene ontology (GO) analysis of biological process for the reverted genes which are either down- or up-regulated in GMA-iHLC -liver support system group. FIG.10C depicts KEGG pathway analysis for the reverted genes which are either down- or up-regulated in GMA- iHLC -liver support system group.

[0027] FIG.11A depicts a schematic diagram of the generation of iHLC which inducibly expresses anti-apoptotic genes BCL2 and BCL2L1. The HLA-TKO iPSCs were first transduced with lentivirus to carry a doxycycline (Dox)-inducible expression cassette of BCL2 or BCL2L1. The iPSCs were then differentiated into iHLC, followed by the treatment with Dox to induce the overexpression of BCL2 or BCL2L1. FIG.11B depicts RT-qPCR analysis of BCL2 and BCL2L1 expression in HLA-TKO iPSCs with or without Dox treatment (100 ng / ml) for 48 hours. Average ± SD (n=3). FIG.11C depicts images of whole mount immunostaining of iHLCs overexpressing BCL2 (iHLC+BCL2) or BCL2L1 (iHLC+BCL2L1) with E-cadherin and HNF4a antibodies combined with Hoechst 33342 and phallodin to stain nuclei and F-actin, respectively. FIG.11D depicts a graph of albumin secretion of iHLC+BCL2 and iHLC+BCL2L1 measured by ELISA. Average ± SD (n=6). FIG.11E depicts whole mount immunostaining of iHLC overexpressing BCL2 and GFP control with cleaved caspase-3 antibody to detect apoptotic cells. The organoids were treated with or without TNFa (10 ng / ml) and cycloheximide (CHX, 10 mg / ml) for 2 hours. Bar, 200 mm. Percentages of cells staining positive for cleaved caspase-3 are indicate in the images.

[0028] FIG.12A depicts a schematic diagram depicting the configuration of BCL2 / BCL2-overexpressing HLA-TKO iHLC-populated liver support system to post hepatectomy liver failure rat. FIG.12B depicts a time course detailing 80% hepatectomy (HTx) followed by extracorporeal life support (ECLS) of iHLC-liver support system . FIG. 12C depicts a Kaplan-Meier survival curve of HTx rats treated with empty column and BCL2 / BCL2L1-overexpressing iHLC columns. *P=0.031, log-rank (Mantel-Cox) test. FIG. 12D depicts graphs showing time-course changes in plasma levels of bilirubin (Bil), ammonia (NH3), AST and ALT post HTx of rats. Average ± SD. iHLC-BCL2 mitigates increase in liver enzyme levels. FIG.12E depicts representative images of H&E staining of 8 IPTS / 128597941.1Attorney Docket No. KNZ-001WO remnant liver histology of HTx rats treated with empty column- liver support system and iHLC+BCL2L1 liver support system treated groups (Post Operative Day 5; POD5). DETAILED DESCRIPTION

[0029] Severe liver failure is characterized by a systemic inflammatory response and rapid deterioration of functioning of other key vital organs, leading to mortality rates of >50%. There are currently no therapies to recapitulate the central role of the liver in detoxification of metabolites and synthesis of ~90% of the secreted human proteome to support healthy physiology. A liver support system disclosed herein provides a novel solution for the prevention and treatment of severe liver failure based on an ex vivo application that enhances detoxification, regeneration, and hepatic synthetic function with optimized induced hepatocyte-like cells (iHLC) and controls inflammation by granulocyte apheresis. Liver Support Systems

[0030] Provided herein are liver support systems comprising (a) an apheresis column; and (b) a detoxification column comprising a plurality of liver tissues, wherein the liver support system is configured to allow a fluid to flow from the apheresis column to the detoxification column. A liver support system disclosed herein can be a tandem-type combination BAL system that utilizes gene-edited, optimized liver cells derived from induced pluripotent stem cells (iPSCs) with excellent synthetic and detoxification functions. It can be used extracorporeally and enhance immune homeostasis through granulocyte and monocyte adsorption, further improving the survival capacity of iHLCs. In some embodiments, the liver support system is configured to allow a fluid to flow from the apheresis column to the detoxification column. The apheresis column can function to reduce immune-induced cytotoxicity of liver cells or tissues present in the detoxification column, e.g. by reducing the activity of or removing immune cells from the fluid. In some embodiments, the apheresis column reduces toxicity of the liver cells or tissues. In some embodiments, the apheresis column comprises one or more modifications for enhanced leukoadsorption. In some embodiments, the apheresis column is configured to adsorb one or both of granulocytes and monocytes from the fluid. In some embodiments, the apheresis column is configured to adsorb granulocytes from the fluid. In some embodiments, the 9 IPTS / 128597941.1Attorney Docket No. KNZ-001WO apheresis column is configured to adsorb monocytes from the fluid. In some embodiments, the apheresis column is configured to adsorb granulocytes and monocytes from the fluid.

[0031] In some embodiments, the apheresis column is a granulocyte and monocyte absorption apheresis (GMA) column. GMA columns can function by selectively depleting activated granulocytes and monocytes / macrophages from a fluid via binding of proteins expressed on the surface of the cells. Removal of immune cells from the fluid can function to not only remove effector cells, but also to remove pro-inflammatory cytokines from the fluid. Use of a GMA column in a liver support system can therefore protect a subject undergoing treatment with the liver support system from systemic toxicity. Use of the GMA column in a liver support system can also protect liver cells or tissues in the detoxification column from inflammatory cells or molecules in the fluid and enhance the function of liver cells or tissues in the detoxification column, e.g., by reducing inflammation of the liver cells or tissues. In some embodiments, the GMA column comprises cellulose acetate beads.

[0032] The detoxification column can provide detoxification, secretory and regenerative function(s). Detoxification can include promoting the central urea cycle which is severely compromised and CYP function which is used for detoxification of xenobiotics, cellular metabolism and homeostasis. Secretory function can include providing proteins and / or metabolites for maintaining vital organ function. Regenerative function can include promoting regeneration of damaged liver The apheresis column can function to adsorb granulocytes from the fluid and to protect the plurality of liver tissues from inflammatory damage.

[0033] In some embodiments, the detoxification column is configured to remove toxic substances from the fluid, provide factors that maintain liver function, and / or to promote regeneration of a damaged liver. In some embodiments, the fluid is whole blood. In some embodiments, wherein the blood is human blood. In some embodiments, the fluid is plasma.

[0034] In some embodiments, the device further comprises a plasma separator between the apheresis column and the detoxification column. In some embodiments, the system comprises one or more modifications to reduce mast cell activity.

[0035] In some embodiments, the liver support system is configured for the fluid to flow at a rate of 30-600 mL / hr. In some embodiments, the liver support system comprises a peristaltic pump. In some embodiments, the liver support system is configured to allow to fluid to flow (1) from a subject, (2) to the apheresis column, (3) to the detoxification column, and (4) back to the subject. 10 IPTS / 128597941.1Attorney Docket No. KNZ-001WO

[0036] In some embodiments, a subject’s blood is flown through the liver support system for a period of at least two hours, at least three hours, at least six hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 38 hours, at least 42 hours, or at least 48 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least two hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least three hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least six hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 12 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 18 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 24 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 30 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 36 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 38 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 42 hours. In some embodiments, a subject’s blood is flown through the liver support system for a period of at least 48 hours.

[0037] Advantages of the liver support system disclosed herein, comprising two tandem circuits, can include: ^ Superior cell source and quality: Gene-edited stem cells that minimize immunogenicity (e.g., MHC-I, II depletion), maximize resistance to oxidative stress (e.g., active gulonolactone oxidase), and optimize synthetic and detoxification functions compared to ELAD, which used immortalized cells. ^ Improvement of immune homeostasis: Granulocyte apheresis is a clinically relevant leukocyte adsorption device currently used for managing systemic inflammation including that due to severe alcoholic hepatitis and inflammatory bowel disease. By directly adsorbing leukocytes, this approach can suppress systemic inflammation, which is a pathogenic condition in ACLF / ALF, and further protects iHLC, thereby enhancing cell survival rates. ^ Simplicity: a liver support system disclosed herein can be optimized for clinical use and complement standard treatments. It can utilize conventional venous access and flow rates to minimize device failures, which are issues with MARS. 11 IPTS / 128597941.1Attorney Docket No. KNZ-001WO Unlike ELAD or other emerging BAL devices using HiHep or Spheroid reactor, a liver support system disclosed herein does not necessarily use a plasma separation device. ^ Compatibility with standard treatments: a liver support system disclosed herein can allow for combination therapies such as with terlipressin, blood filtration dialysis, and plasma exchange.

[0038] Also provided are: ^ Novel engineering design(s) of a tandem circuit that includes the sequential use of granulocyte apheresis and iHLC in a column to enable single or multiple use application for up to 48 hours per session. ^ The granulocyte apheresis can result in reduction in the counts of monocytes and neutrophils, as well as levels of cellular immune markers of inflammation, cytokines, and chemokines. This approach can serve to reduce systemic inflammation and / or protect the iHLC from immune-mediated cellular injury. ^ Cell source: iHLC are induced pluripotent stem cell (iPSC)-derived human hepatocyte-like cells that can have the following one or more modifications, including: o Reduced immune response (T-cell: class I and II edits; NK-cell: HLA- E knock-in, CD47 knock-in; innate: NLRP3 knock-down, IL1b knock-in) o Enhanced tolerance to oxidative stress (edits to knock-in mGULO, NRF2, MSRP5) o Improved functionality (edits to enhance albumin affinity and functionality, urea cycle enzymes, knock-out ACMSD, CD38 / CD73) o Improved regenerative potential of the native or transplanted liver (HNF4, TBX3, FOXA2, FOXA1, PROX1, HNF1B, GATA4, NANOG, SOX2, OCT3 / 4, c-MYC). These genetic modifications to the iHLC will typically be either inducible or provide constitutive expression. Additional cell sources include primary hepatocytes, primary liver organoids, primary ductal organoids, fetal hepatocytes, hepatic progenitors, hepatoblasts, iPSC / ESC-derived hepatocytes, directly reprogrammed hepatocytes (hiHep). Different forms of iHLC include 2D, 3D, aggregate, tissue slice, or organoids. 12 IPTS / 128597941.1Attorney Docket No. KNZ-001WO ^ Adaptation to whole blood circuit without need for plasma separation. This can use the agranulocyte apheresis column, which leads to a reduction in polymorphonuclear cell count and activation, and lack of enhanced coagulation in the iHLC column. Options can include flow of whole blood through both columns; alternatively, whole blood for granulocyte apheresis, followed by plasma separation for flow through iHLC column. ^ Convenient venous access – both central and peripheral – with flow rates of, e.g., 30-200 mL / hr to optimize for continuous use.

[0039] Liver support systems disclosed herein are highly differentiated from other efforts. Key features of differentiation can include: ^ Initial immune modulation using granulocyte apheresis and / or immune adsorption column ^ Genetically-edited iHLC for superior functionality in terms of detoxification, synthetic function, regenerative potential ^ Ease of use based on convenient venous access and flow rates Liver Tissues and Cells

[0040] In some embodiments, liver tissues comprise cells such as pluripotent stem cell- derived induced hepatocyte-like cells aggregates or organoids (iHLCs), liver bud organoids, liver organoids, primary hepatocytes, primary liver organoids, primary ductal organoids, fetal hepatocytes, hepatic progenitors, hepatoblasts, stem cell-derived hepatocytes, and directly reprogrammed hepatocytes (hiHep).

[0041] In some embodiments, liver tissues comprise engineered hepatocyte-like cells. In some embodiments, liver tissues comprise iHLCs. In some embodiments, the iHLCs are engineered human cells, optionally induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the iHLCs comprise one or more genetic alterations to confer resistance to oxidative stress.

[0042] Table 1 provides a list of modifications to the apheresis column or gene edits for iHLC production. The goal of these edits is to produce durable cells with minimal loss of functional activity under heterogenous clinical conditions. Universality to minimize cell loss by immune cells / SIRS mediated by T-cells, NK-cells, and activated macrophages can be achieved with use of the granulocyte adsorption column prior to iHLC, and modifications to the column. 13 IPTS / 128597941.1Attorney Docket No. KNZ-001WO Table 1: Column Modifications and Gene Edits for iHLC Category Description / Edits Hypoimmune / Reduced Edits to MHC Class I (e.g., b2M) and / or MHC class II (e.g., i fl i IITA or t

[0043] In some embodiments, the one or more genetic alterations to confer resistance to oxidative stress comprise expression of a constitutively active variant of nuclear factor erythroid 2-related factor 2 (NRF2), mouse L-gluconolactone oxidase (mGULO), mutant albumin domain 2 comprising F211R and R218L amino acid substitutions, or MSRP5. In some embodiments, the iHLCs comprise one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling. In some embodiments, the one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling comprise: (a) edits to MHC class-I and / or MHC class-II genes; (b) suppression of expression of HLA-A, HLA-B, B2M, NLRP3, and / or CIITA; and / or (c) induction of expression of HLA-E and / or anti-IL1b. In some embodiments, the iHLCs comprise one or more genetic alterations to improve longevity of the iHLCs. In some embodiments, the one or more genetic alterations to improve longevity comprise one or more alterations to genes involved in cell cycle, 14 IPTS / 128597941.1Attorney Docket No. KNZ-001WO apoptosis, and / or metabolism. In some embodiments, the one or more alterations to genes involved in metabolism comprise alterations to genes involved in fatty acid oxidation. In some embodiments, the iHLCs comprise one or more genetic alterations to improve detoxification function and / or hepatic synthetic function of the iHLCs. In some embodiments, the one or more genetic alterations to improve detoxification function and / or hepatic synthetic function comprise: (a) one or more genetic alterations to genes involved in albumin affinity, albumin functionality, urea cycle, and / or bilirubin clearance; coagulation factor genes and / or cytochrome P450 genes; and / or (b) suppression of expression of ACMSD, CD38, PARP, and / or CD73. In some embodiments, the iHLCs comprise one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver. In some embodiments, the one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver comprise ectopic expression of HNF4A, TBX3, FOXA1, FOXA2, FOXA3, FOXA1, PROX1, HNF1A, HNF1B, CEBPA, NR1I2, KDM2B, GATA4, HNF6, ATF5, HHEX, NANOG, SOX2, OCT3, OCT4, c-and / or c-MYC, CHIR99021, RepSox, Forskolin, Valproic acid, Parnate, TTNPB, and / or DZNep.

[0044] In some embodiments, the engineered hepatocyte like cells comprise inducible expression of one or more anti-apoptotic proteins. In some embodiments, wherein the anti- apoptotic protein is a BCL2 family protein. In some embodiments, the anti-apoptotic protein is selected from the group consisting of BCL2, BLC2L1 (BCL-XL), BAX, IAP1, BIRC3,XIAP, NAIP, BIRC5, BIRC7, ILP2, and BIRC6. In some embodiments, the anti- apoptotic protein is BCL2. In some embodiments, the anti-apoptotic protein is BLC2L1 (BCL-XL). In some embodiments, expression of the one or more anti-apoptotic protein is under the control of a doxycycline-inducible promoter.

[0045] In some embodiments, the engineered cell has reduced or substantially no expression of one or more protein selected from the group consisting of HLA-A, HLA-B, and CIITA. In some embodiments, the wherein the cell has reduced or substantially no expression of each of HLA-A, HLA-B, and CIITA. In some embodiments, the expression of the one or more proteins is reduced by gene editing. In some embodiments, the expression of the one or more proteins is reduced by CRISPR-Cas9-mediated gene editing.

[0046] In some embodiments, the iHLCs are comprised within a two-dimensional culture, a three-dimensional culture, a cellular aggregate, a tissue section, or one or more liver organoids. In some embodiments, the iHLCs are comprised within one or more liver organoids. 15 IPTS / 128597941.1Attorney Docket No. KNZ-001WO Indications

[0047] Also disclosed herein, in various embodiments, is a method of treating a liver disease in a subject in need thereof, the method comprising flowing whole blood from the subject through the liver support system disclosed herein.

[0048] Contemplated indications include: liver failure associated with or without systemic inflammation wherein a liver support system disclosed herein provides a bridge to recovery and / or liver transplantation. Specific indications include treatment of: ^ Acute liver failure (ALF) due to toxins, medications, vascular injury, infection, and other etiologies, including prevention or treatment of complications of liver mass resection or small for size liver transplant. ^ Acute-on-chronic liver failure (ACLF), including in conjunction with terlipressin and other treatment modalities approved or in development (e.g., NAD+ modulation) for ACLF-1, as well as for ACLF-2,3 in conjunction with standard of care treatments (e.g., continuous renal replacement therapy [CRRT], plasmapheresis). ^ Liver failure due to genetic inborn errors of metabolism, mitochondrial disease, and acute fatty liver of pregnancy. ^ Intractable pruritus, for example, due to chronic cholestatic liver diseases. ^ Prevention or treatment of liver failure or liver injury post liver mass resection ^ Promote liver regeneration post liver mass resection

[0049] Also disclosed herein, in various embodiments, is a method of treating a liver disease in a subject in need thereof, the method comprising flowing whole blood from the subject through the liver support system disclosed herein. In some embodiments, the subject is afflicted with is acute-on-chronic liver failure (ACLF), acute liver failure (ALF), liver failure due to genetic inborn errors of metabolism, liver failure due to mitochondrial disease, acute fatty liver of pregnancy, intractable pruritus, drug-induced liver injury (DILI), or ischemic hepatitis. In some embodiments, the liver disease is ACLF. In some embodiments, the ACLF is ACLF-1, ACLF-2, or ACLF-3.

[0050] In some embodiments, the subject is afflicted with viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis of any etiology, sepsis, or systemic inflammatory response syndrome. In some embodiments, the subject is awaiting liver transplant. In some embodiments, the subject has undergone liver transplant or liver 16 IPTS / 128597941.1Attorney Docket No. KNZ-001WO resection. In some embodiments, the subject is receiving or has received terlipressin, continuous renal replacement therapy, plasmapheresis, blood filtration dialysis, and / or plasma exchange. Efficacy, effects, or performance

[0051] Treatment of patients with liver disorders is characterized by severe liver dysfunction based on advanced liver dysfunction due to factors such as alcohol abuse, infections, gastrointestinal bleeding, and worsening of the underlying disease. MOA

[0052] The iHLC contained in this product, when combined with granulocyte adsorption by Apheresis column, can demonstrate the ability to remove systemic inflammatory response syndrome (SIRS) and accumulated toxic substances, which are the pathologies of ACLF. This action, which exhibits pharmacological effects against acute inflammation and toxic substance accumulation that were difficult to manage with conventional treatments, can contribute to improving prognosis.

[0053] The reduction of inflammatory cytokines through granulocyte adsorption and the removal of toxic substances and replenishment of beneficial substances derived from genetically modified iPSC-derived iHLC to be confirmed. Specifically, significant reduction of the inflammatory cytokine IL-6, increase in hepatocyte growth factor (HGF) that promotes liver regeneration, and substantial enhancement of toxic substance removal through further genetic modification of low immunogenicity iPS cells will have been achieved. With a liver support system disclosed herein treatment, in addition to its anti-inflammatory effects, reduction of bilirubin and ammonia levels, and replenishment of plasma proteins are expected, leading to improved survival rates. EXAMPLES Example 1: Extracorporeal Circulation of Blood using a Liver Support Device

[0054] Intended use (route / site): Jugular vein, femoral vein for catheter placement for dialysis. Dosage: To be determined in animal experiments. Specific Protocol:

[0055] Using drapes, the surgical field is sterilized with povidone-iodine solution or similar disinfectant applied outward from the insertion site. Local anesthesia is applied to the 17 IPTS / 128597941.1Attorney Docket No. KNZ-001WO insertion site. A syringe filled with heparinized saline is attached to a plastic cannula needle and used to puncture the blood vessel. After confirming the absence of backflow, the metal puncture needle is held in place, and only the plastic cannula is advanced and positioned inside the blood vessel. A guidewire is threaded through the inserted cannula into the blood vessel. The cannula is removed, and a small surgical incision is made at the insertion site using a scalpel. A dilator is guided along the guidewire, gradually advancing while confirming resistance, sufficiently expanding the subcutaneous tissue and the entry point to the blood vessel, and then the dilator is removed. A catheter is slowly inserted along the guidewire. The depth scale is checked at every 1 cm while advancing. Confirmation of proper placement of the catheter in the desired position, followed by removal of the guidewire and stylet. After removing any air from the blood delivery and blood removal catheters, the catheters are flushed with heparinized saline and clamped. The detachable catheter skin fixation device is secured with sutures. The blood removal side of the catheter is connected to the blood removal side of the extracorporeal circulation circuit, which includes iHLC and Apheresis beads -containing columns, and the blood return side is connected to the blood return side of the extracorporeal circulation circuit. The extracorporeal circulation is initiated. After 3 hours of extracorporeal circulation, the blood removal catheter is flushed with heparinized saline, locked with heparin, the adapter is thoroughly disinfected, and an infusion plug is attached. The blood delivery side is subjected to the same procedure. Example 2: Ex vivo demonstration of tandem circuit protection of the iHLC column.

[0056] Viability of iHLC will be measured after incubating for 1h with rat blood directly, or post flow-through in the granulocyte adsorption column. We predict near total loss of viability of iHLC following direct exposure versus >80% viability with use of the proposed tandem sequential circuit. Example 3: Ex vivo demonstration of enhanced functionality, synthetic function, and detoxification with edited iHLC.

[0057] We will assess albumin and coagulation factor levels in immune edited iHLC with mGULO and / or NRF2 knock-in, and other edits. We predict higher levels of albumin and factor V, VII and IX in edited cells. We will assess reductions in bilirubin levels following a 2h incubation of iHLC with serum from patients with hyperbilirubinemia. We predict a >20% reduction in serum bilirubin concentrations. Example 4: In vivo demonstration of efficacy in rodent model of ACLF. 18 IPTS / 128597941.1Attorney Docket No. KNZ-001WO

[0058] We will use an established model wherein rats are subjected to bile duct ligation and administered lipopolysaccharide (LPS), which is reflective of sepsis associated with liver failure. These rats develop progressive liver fibrosis by 3-4 weeks and are highly susceptible to LPS administration with 80-100% mortality within 48h. We will evaluate the tandem sequential a liver support system disclosed herein in a controlled study design. We predict near universal (80-100%) mortality in untreated rats compared to 0% mortality in rats treated with the device / iHLC approach. Example 5: Ex vivo demonstration of tandem circuit to improve cell survival Methods CRISPR-Cas9 Gene Editing

[0059] The Ff-I01s04-AbⅡ KO was generated from the human iPSC line 1383D6 via CRISPR-Cas9- mediated knockout of HLA-A, HLA-B, and CIITA genes as described in Xu H, et al. Cell Stem Cell.2019 Apr 4;24(4):566-578.e7. Briefly, gRNA candidates were selected from the exon 2-3 regions of the target gene alleles and targetability of the selected gRNA sequences was confirmed using WGS or WES data. Potential off-target loci of the selected gRNAs was assessed using CRISPOR (crispor.tefor.net) or GGGenome (gggenome.dbcls.jp) tools. gRNAs were prepared by in vitro transcription using a MEGAshortscript kit (Thermo Fisher Scientific) according to the instruction manual. Transfection was performed using 4D-Nucleofector (Lonza).0.3 × 106iPSCs were transfected with a RNP (ribonucleoprotein) complex of 5 μg recombinant Cas9 (IDT, Cat. No.1074181 or ThermoFisher, Cat. No. A36498) and 1.25 μg of IVT gRNA in 20 μL Primary P4 buffer and the electroporation condition program CA-137. After electroporation, cells were transferred to 0.48 μg / cm2 iMatrix-511-coated 6-well plates and grown in AK03N medium containing ROCK inhibitor Y-27632. All iPSC lines were cultured with StemFit AK02N (Ajinomoto) and iMatrix-511 (Nippi) at 37°C in 5% CO2with 95% air. Generation of iHLCs

[0060] Human iPSCs were differentiated into posterior gut endoderm progenitors (EP) as described in Zhang et al. “Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids.” Stem Cell Reports.2018 Mar 13;10(3):780-793. In brief, human iPSCs were detached by Accutase (Thermo Fisher Scientific Inc.) and were seeded on iMatrix-511-coated tissue culture plates with 100,000 19 IPTS / 128597941.1Attorney Docket No. KNZ-001WO cells / cm2. Medium was changed to RPMI 1640 medium supplemented with 1× non-essential amino acids (Thermo Fisher Scientific Inc.) that contain 100 ng / mL activin A (R&D Systems), 50 ng / mL bone morphogenetic protein 4 (BMP4; R&D Systems), and 0.25% B27 (Gibco) at day 1, 100 ng / mL activin A and 0.2% fetal bovine serum (Thermo Fisher Scientific Inc.) at day 2, and 100 ng / mL activin A and 2% fetal calf serum at day 3. On days 4 to 6, cells were cultured in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) with 1× B27 (Life Technologies) and 1× N2 (Gibco) containing 500 ng / mL FGF4 (R&D Systems) and 3 μM CHIR99021 (Stemgent) to induce the FG. The medium was replaced every day. The obtained FG cells were detached by Accutase and then frozen in CELLBANKER 1 freezing medium (Nippon Zenyaku Kogyo Co, Ltd), and stored in -80°C or in liquid nitrogen for long-term storage.

[0061] The EP cells were quickly thawed and then centrifuged at 300 ×g for 5 minutes. Cells were resuspended in Matrigel (Corning, Inc) on ice with approximately 100,000 viable cells being embedded in 50 µL Matrigel drops, and subsequently grown in Advanced DMEM / F12 with 2% B27, 1% N2, 1× HEPES, 1× GlutaMAX, 1% Pen / Strep, 3 μM CHIR99021, 20 ng / ml EGF, 1 μM A83-01, 10 ng / ml VEGFP, 5 ng / ml bFGF, 25 ng / ml IGF1, and 50 μg / ml ascorbic acid. Following the culture with Y-27632 for 3 days and additional 2- 3 days without Y-27632 to form the spheroids, the medium was replaced with Advanced DMEM / F12 with 2% B27, 1% N2, 1× HEPES, 1× GlutaMAX, 1% Pen / Strep, 2μM A83-01, and 1 μM A83-01 for 2 days and the medium without A83-01 for further 2 days with medium changed every other day. The generated organoids were isolated from Matrigel and then cultured in the suspension culture in HCM Hepatocyte Culture Medium BulletKit (Lonza; human EGF singlequot was removed) supplemented with 100 nM dexamethasone, 20 ng / ml Oncostatin M, 10 ng / ml HGF, 10 μM DAPT, and 10% Matrigel at the ultra-low attachment 6-well plate (Thermo Fisher Scientific Inc.) for 10-14 days, with medium changed every 3 day. Encapsulation of iHLC into Alginate

[0062] Sodium alginate powder (Wako Pure Chemical, Cat # 194-13325) was dissolved in water to a concentration of 1.5% (w / v) and filter sterilized through a 0.22 µm filter. iHLCs were collected and centrifuged at 300 ×g for 5 min at 4°C. Next, the supernatant was removed and the iHLC pellet was gently disrupted by tapping the tube. The iHLCs were resuspended in the alginate solution at a density of approximately 4×103organoids per ml. 20 IPTS / 128597941.1Attorney Docket No. KNZ-001WO Droplets (approximately 30 uL per droplet) of the alginate containing iHLCs were directly dropped into 0.4% BaCl2solution and then allowed to polymerize for 15 min at room temperature. The microcapsules were washed with PBS three times to remove excess Ba2+ions. The encapsulated iHLCs were maintained in HCM medium until they were used for liver support system experiments. Flow Cytometry Analysis of iHLC

[0063] To analyze the surface expression of HLA, iHLCs were dislodged from Matrigel and transferred into a low-attachment 6-well plate followed by treatment with or without 25 ng / ml IFN-γ (Proteintech) for 24 hr. The organoids were then collected and treated on ice with Cell Recovery Solution (Corning) for 10 min to dissolve the remaining Matrigel. They were collected in the wash buffer (DMEM / F12 supplemented with 0.1% BSA fraction V) and further digested into single cells using TrypLE Express Enzyme (Gibco) for 15-20 min at 37°C water bath. The dissociated cells were collected in wash buffer and centrifuged at 300 ×g for 10 min. Each cell pellet was resuspended in Cell Staining Buffer (BioLegend) with Ghost Dye Violet 450 (TONBO). For staining, the cell suspension was incubated with 100 μl of diluted primary antibody for 30 min on ice. Anti-HLA-A24 mAb-Alexa Fluor 647 (MBL) was used for HLA-A staining. Anti-HLA-B7 antibody(Bio-Rad) followed by secondary staining with Goat Alexa Fluor 647 anti-mouse IgG antibody (Invitrogen) was used for HLA- B staining. After washing, samples were analyzed on a BD LSRFortessa (BD Biosciences), and the data were processed using FlowJo software (Ver.10.10.0). Immunofluorescence

[0064] Encapsulated iHLCs were fixed in 4% paraformaldehyde at 4°C overnight. iHLCs were washed with PBS, 1mM EDTA was added, pipetted and centrifuged at 300 g for 5 min to remove the supernatant. The following primary antibodies were used: caspase-3 antibody (Cell Signaling Technology), E-cadherin antibody (R&D), and HNF4a antibody (Abcam). Samples were washed 3 times with washing buffer (0.1% Triton-X 100 / PBS) followed by secondary labelling with Alexa Fluor-conjugated donkey anti-rabbit / mouse / goat immunoglobulin G (Life Technologies). After the secondary reaction, the samples were washed 3 times with washing buffer. Hoechst 33342 and rhodamine-conjugated phalloidin were used to counterstain nuclei and F-actin, respectively. TUNEL assay kit (Cell Signaling Technology) was used to detect the fragmented DNA. The stained samples were visualized and scanned on KEYENCE BZ-X800 Fluorescence Microscope. 21 IPTS / 128597941.1Attorney Docket No. KNZ-001WO Liver Support System

[0065] 1-mL columns were filled with 300 µm Adacolumn granulocyte and monocyte adsorption apheresis (GMA) cellulose acetate beads (JIMRO) for the apheresis column or iHLC alginate capsules (6-10 capsules) for the detoxification column. Each column was placed in series and a circuit was assembled. The circuit was connected to a peristaltic pump and the tubing and column were filled with heparinized saline. 26G intravenous catheters were placed in the left and right external jugular veins of rats under continuous anesthesia by isoflurane. The catheters were connected to the circuit. Statistical Analysis

[0066] Data were presented as means ± SD. An unpaired two-tailed Student’s t-test and analysis of variance (ANOVA) were used to calculate statistical significance. For survival time, the Mantel-Cox log-rank test was performed. P < 0.05 was considered statistically significant. Statistical calculation was performed using GraphPad Prism 7. Results

[0067] Induced hepatocyte-like cells (iHLCs) were efficiently generated from induced pluripotent stem cells (iPSCs) via a posterior gut endoderm progenitor intermediate (FIG. 2A). iHLCs derived from HLA triple knockout or wild-type iPSCs were cultured in Matrigel and imaged (FIG.2B), showing a morphology of the triple-knockout iHLC compared to iHLCs generated from wild-type cells. To assess immunoreactivity, iHLCs were analyzed for HLA-A and HLA-B surface expression by flow cytometry (FIG.2C). The absence of HLA upregulation confirms the hypoimmune phenotype of the triple knockout.

[0068] iHLCs were next dissociated from Matrigel and encapsulated in alginate (FOG. 3A). Cells encapsulated in alginate or cultured in Matrigel were imaged for markers of live and dead cells (FIG.3B). Images showed that alginate encapsulation maintained the viability of iHLCs in vitro. Albumin secretion from iHLCs was measured to assess whether the cells exhibit normal hepatocyte cell function (FIG.3C), showing similar levels of albumin secretion in alginate encapsulated iHLCs compared to Matrigel culture.

[0069] The viability of encapsulated spheroids of HepG2 human hepatoma cells, which have been previously used in bioartificial liver devices, housed in a liver support system was assessed following extracorporeal circulation of a rat. The system, including a GMA column, was connected in tandem with the HepG2 column in two different configurations: GMA 22 IPTS / 128597941.1Attorney Docket No. KNZ-001WO column-to-HepG2 column and HepG2 column-to-GMA column. Two hours after extracorporeal circulation in healthy rat, HepG2 spheroids were collected and staining with calcein-AM / EthD-1 (FIG.4). Maximal cell viability (middle panel) was observed with the GMA column-to-HepG2 column configuration.

[0070] The viability of encapsulated iHLC following extracorporeal circulation of an acute-on-chronic liver failure (ACLF) rat model (FIG.5A) was assessed. Rats underwent bile duct ligation (BDL) to induce cirrhotic liver condition, followed by intraperitoneal LPS injection as a second, acute trigger. One hour after LPS injection, rats received the treatment of a liver support system with an encapsulated iHLC column in the presence or absence of GMA column (upstream of the iHLC column) for two hours. After the treatment, iHLCs were collected, followed by whole mount staining of cleaved caspase-3 and F-actin. Encapsulated iHLCs without extracorporeal circulation were used as a control. Percentages of cells staining positive for cleaved caspase-3 were quantified (FIG.5B) to assess apoptosis of iHLCs. Human mitochondrial DNA (mtDNA) was detected in the rat serum following extracorporeal circulation to detect evidence of cell death of hepatocytes in the column. (FIG.5C). The data in this inflammatory model of ACLF demonstrate that the GMA column significantly reduces apoptotic cell death of iHLC in the system. Example 6: Superior in vivo efficacy of tandem circuit containing HLA-knockout iPSCs Methods Liver Support System Treatment in Rats Liver support system circuits were primed with heparinized saline (10 units / ml heparin), followed by a bolus dose of 200 units of heparin via catheter at the start. Rats were divided into groups for treatment with empty columns or GMA-iHLC liver support system. In the empty column and GMA-iHLC groups, LPS was administered for 1 hour, followed by extracorporeal circulation. Sedation was provided with 2% isoflurane during treatment. Rats were connected to an extracorporeal device via left and right jugular vein catheters. Peristaltic pumps were set at a flow rate in 1 mL / min. The rats underwent 2 hours of extracorporeal circulation. After liver support system treatment, all rats were awakened, caged and followed up for 3 days. Blood samples were collected before and after liver support system treatment. Plasma toxin (bilirubin and ammonia) and inflammatory cytokine 23 IPTS / 128597941.1Attorney Docket No. KNZ-001WO levels were detected. Every day afterward, blood was sampled until the animals were euthanized at the endpoint.

[0071] Coma severity was assessed at 3 and 24 hours after treatment according to the coma scaling for rats. The following reflexes, physiological responses and behavioral domains were assessed. Motor function (reflexivity, purposefulness), brainstem reflexes (pupillary, corneal and fin reflexes), breathing, righting reflexes, auditory responses and whisker movements. Immunohistochemistry

[0072] Liver samples were fixed overnight in 4% PFA and embedded in paraffin blocks. The following primary antibodies were used: Ki67 antibody (Abcam), p21 antibody (Invitrogen), and MPO antibody (Abcam). Samples were washed 3 times with washing buffer (0.1% Triton-X 100 / PBS) followed by secondary labelling with Alexa Fluor conjugated anti-mouse / -rat / -rabbit IgG antibody (Invitrogen).After the secondary reaction, the samples were washed 3 times with washing buffer. The stained samples were visualized using Keyence fluorescence microspce. RNA-seq

[0073] Total RNA was extracted from snap-frozen rat liver tissues by using a two-step protocol. Briefly, the frozen tissues were homogenized in TRI reagent (Molecular Research Center, Inc.) followed by addition of chloroform and mixing for phase separation. After centrifugation, the aqueous phase was isolated, which was further re-extracted with FastGene RNA Premium Kit (NIPPON Genetics, Co., Ltd.) including Dnase I treatment. The RNA- seq libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext UltraTMII Directional RNA Library Prep Kit (New England Biolabs). Subsequently the libraries were sequenced using a paired-end 150-bp protocol on an Illumina NovaSeq 6000. The quality of the raw paired-end sequence reads was assessed with FastQC (v0.11.7). Low quality and adapter sequences were trimmed by Trimmomatic (v0.38). The trimmed reads were aligned to the reference genome (rn6) with HISAT2 (v2.1.0) with default parameters. The bam files were used to estimate the abundance of uniquely mapped reads with featureCounts (version 1.6.3). The raw read counts were normalized and processed for differential gene expression analysis using DESeq2 (v1.24.0) in R software. Statistical significance of genes was determined by an adjusted p <0.05 and |fold change| > 2. Gene 24 IPTS / 128597941.1Attorney Docket No. KNZ-001WO ontology analysis was performed on the differentially expressed genes using enrichGo in the clusterProfiler R package (v4.10.0) and Enrichr. Results

[0074] ACLF rats underwent bile duct ligation and were then injected with LPS and connected to a liver support system comprising a GMA and / or iHLC column connected in tandem and underwent extracorporeal circulation for two hours, rats were then monitored for 3 days with blood sampling every 24 hours. (FIG.6A). Rats were monitored for survival (FIG.6B) and plasma levels of ammonia, AST, ALT, and total bilirubin (FIG.6C). Only the tandem application of GMA-iHLC provided clinical benefit with a significant improvement in survival. The individual components of the GMA-iHLC circuit showed no significant survival benefit. In addition to survival benefit, significant improvement in laboratory parameters of liver function and liver pathology were observed. Coma severity (n=2 each) was assessed at 3 and 24 hours after the liver support system treatment according to the coma scoring for rats including eye blink, motor function, brainstem reflexes, respiration, righting reflex, auditory response, and whisker movement (FIG.6D and Table 2). The GMA-iHLC column mitigated ACLF related coma severity as early as 3 hours and the benefits were maintained through 24 hours. Table 2: Coma Severity of Rats Following Bile Duct Ligation and Extracorporeal Circulation GMA-iHLC Tandem Circuit Empty Column25 IPTS / 128597941.1Attorney Docket No. KNZ-001WO

[0075] Liver samples were collected from rats having undergone BDL and extracorporeal circulation with a GMA-iHLC liver support system or an empty column control and assessed for Ki67 staining by immunohistochemistry (FIG.7A-7B). The increase in Ki67+ cells indicated greater liver regeneration in animals treated with the GMA-iHLC liver support system. Liver samples from rats 24 hours following extracorporeal circulation were collected and stained for p21 by immunohistochemistry (FIG.7C). The observed increase in p21 staining following treatment with the GMA-iHLC liver support system indicated mitigation of senescence and induction of regenerative response.

[0076] RNA sequencing was performed on liver samples from rats having undergone BDL and extracorporeal circulation with a GMA-iHLC liver support system or an empty column control. A large liver transcriptional response was observed in GMA-iHLC liver support system-treated animals (FIG.8A). Gene set enrichment analysis of upregulated genes in the GMA-iHLC-treated group indicated that the increase in transcriptional response was consistent with greater liver regeneration (FIG.8B). Enrichment analysis of ENCODE and ChEA consensus transcription factor and transcription factor perturbation gene sets for upregulated genes in GMA-iHLC liver support system-treated animals reflects the physiological enhancement of HNF4a, the major regenerative pathway in humans (FIG.8C).

[0077] Liver samples were collected from rats having undergone BDL and extracorporeal circulation with a GMA-iHLC liver support system or an empty column control and assessed for MPO staining by immunohistochemistry (FIG.9A). Liver injuries were scored as described in Glorioso JM et al. J Hepatol.2015 Aug;63(2):388-98.(FIG.9B). Animals treated with GMA-iHLC had reduced liver injury markers at day 1 and day 3. Liver samples were imaged for cleaved caspase-3 and TUNEL to detect hepatic toxicity in rats treated with GMA-iHLC or control (FIG.9C). These results indicate reduced toxicity in rats treated with the GMA-iHLC liver support system.

[0078] Gene expression analysis was carried out to further characterize the effects of extracorporeal circulation with the GMA-iHLC liver support system (FIG.10A). Functions of genes which are either up-regulated or down-regulated in rats treated with the GMA-iHLC liver support system were analyzed by gene ontology (FIG.10B) and KEGG pathway analysis (FIG.10C). These results indicated that in the livers of animals treated with GMA- iHLC, there was significant down-regulation of pathways associated with inflammation and 26 IPTS / 128597941.1Attorney Docket No. KNZ-001WO up-regulation of physiological metabolic pathways, which were indicative of rapid restoration of native liver function. Example 7: Superior in vitro characteristics and in vivo efficacy of genetically-edited HLA KO iPSC Methods Preparation of Lentivirus

[0079] Self-inactivating lentiviral vectors that carry a gene encoding human BCL2 or BCL2L1 under the control of a doxycycline-inducible promoter and a gene conferring resistance to puromycin as a selection marker (Masaki et al., Cell Stem Cell 2016) were transiently transfected together with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev (RIKEN BRC) using TransIT-2020 reagent (TaKaRa Bio Inc.) into HEK293T cells. Medium containing lentivirus was collected, and lentiviral infection was conducted by adding the virus-containing medium supplemented with 8 μg / ml of polybrene into the HLA-TKO human iPSCs. After incubation for 1 day, differentiation medium was replaced followed by removal of uninfected cells by puromycin from day 2. 80% Hepatectomy and Extracorporeal Circulation Treatment in Rats

[0080] As a preoperative preparation, rats were fasted on the night before surgery. Anesthesia was induced with 3%-4% isoflurane inhalation and maintained at 2.0-3.0% during the surgery. The partial hepatectomy involved the resection of the median, left lateral, and caudate lobes. Following surgery, rats were administered 12.5 mL / kg of saline subcutaneously and monitored until they fully regained consciousness. After surgery, the animals were kept warm and monitored in a separate cage until they were fully conscious. All procedures were designed to minimize animal suffering.

[0081] Before extracorporeal circulation treatment, rats with 80% hepatectomy were allowed free access to water and food and were monitored. Following this observation period, they were then randomly divided into the treatment groups. The GMA-iHLC circuit was primed with heparinized saline (10 units / ml heparin), followed by a bolus dose of 200 units of heparin via catheter at the start. Rats were connected to an extracorporeal device, where iHLC column was connected to the air trap, via internal jugular artery and femoral vein catheters. Peristaltic pumps were set at a flow rate in 1 ml / min. The rats underwent 2 h of extracorporeal circulation. Blood samples were collected at regular intervals before 27 IPTS / 128597941.1Attorney Docket No. KNZ-001WO starting ECLS treatment, immediately after ECLS treatment, 48 hours post-hepatectomy, and at the predetermined endpoint post-hepatectomy. Serum levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Total Bilirubin (T-Bil), and Ammonia were monitored. The postoperative day 5 is set the primary endpoint of this study.

[0082] Rats with more than 1 ml of blood loss were excluded. All animals that reached the status of “death equivalence” were euthanized according to an ethical policy of laboratory animals. Results

[0083] Triple-knockout iHLCs were further engineered to inducibly express anti- apoptotic genes BCL2 (iHLC-BCL2) or BCL2L1 (iHLC-BCL2L1) (FIGs.11A-11B). Engineered cells were cultured in Matrigel and stained for E-cadherin and HNF4a (FIG. 11C). Engineered cells were further assessed for albumin secretion (FIG.11D), indicating that iHLC-BCL2 cells maintain liver function and albumin production and are superior to iHLC without BCL2 expression. Engineered cells were further cultured in Matrigel, treated with or without TNFα or cycloheximide (CHX) and stained for cleaved caspase-3 to detect apoptotic cells (FIG.11E). These results indicate that iHLC-BCL2 are protected against TNFα / CHX induced apoptosis.

[0084] To assess the effects of iHLC-BCL2 and iHLC-BCL2L1 cells in a liver support system on liver function, iHLC-BCL2 and iHLC-BCL2L1 cells were seeded into columns and connected to a liver support system in tandem with a GMA column. Rats were subjected to an 80% hepatectomy (HTx) and connected to the iHLC-BCL2 or iHLC-BCL2L1 liver support system (FIG.12A). Rats underwent extracorporeal circulation for two hours, followed by blood sampling over 72 hours, and were then euthanized for blood / liver sampling (FIG.12B). Rats mere monitored for survival (FIG.12C), which indicated that the iHLC-BCL2 liver support system provided significantly higher survival rates. Rats were further monitored for plasma levels of bilirubin (Bil), ammonia (NH3), AST and ALT (FIG. 12D), indicating that the iHLC-BCL2 liver support system mitigated an increase in liver enzyme levels. Liver samples were collected from rats and analyzed by H&E staining (FIG. 12E), indicating that the iHLC-BCL2 liver support system provided more rapid recovery of the native liver. INCORPORATION BY REFERENCE 28 IPTS / 128597941.1Attorney Docket No. KNZ-001WO

[0085] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0086] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. 29 IPTS / 128597941.1

Claims

Attorney Docket No. KNZ-001WO WHAT IS CLAIMED IS: A liver support system comprising, in sequence: an apheresis column; and a detoxification column comprising a plurality of liver cells or tissues, wherein the liver support system is configured to allow a fluid to flow from the apheresis column to the detoxification column. The liver support system of claim 1, wherein the apheresis column is configured to adsorb one or both of granulocytes and monocytes from the fluid. The liver support system of claim 1 or 2, wherein the apheresis column is a granulocyte and monocyte adsorption apheresis (GMA) column. The liver support system of claim 3, wherein the GMA column comprises cellulose acetate. The liver support system of any one of claims 1-4, wherein the apheresis column reduces toxicity of the liver cells or tissues. The liver support system of any one of claims 1-5, wherein the apheresis column comprises one or more modifications for enhanced leukoadsorption. The liver support system of any one of claims 1-6, wherein the detoxification column is configured to remove toxic substances from the fluid, provide factors that maintain liver function, and / or promote regeneration of damaged liver. The liver support system of any one of claims 1-7, wherein the fluid is whole blood. The liver support system of claim 8, wherein the blood is human blood. The liver support system of any one of claims 1-7, wherein the fluid is plasma. The liver support system of any one of claims 1-10, wherein the device further comprises a plasma separator between the apheresis column and the detoxification column. 30 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The liver support system of any one of claims 1-11, wherein the liver cells or tissues comprise cells selected from the group consisting of: pluripotent stem cell-derived induced hepatocyte-like cell aggregates or organoids (iHLCs), liver bud organoids, liver organoids, primary hepatocytes, primary liver organoids, primary ductal organoids, fetal hepatocytes, hepatic progenitors, hepatoblasts, stem cell-derived hepatocytes, and directly reprogrammed hepatocytes (hiHep). The liver support system of claim 12, wherein the liver cells or tissues comprise iHLCs. The liver support system of claim 13, wherein the iHLCs are engineered human cells, optionally induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). The liver support system of claim 13 or 14, wherein the iHLCs comprise one or more genetic alterations to confer resistance to oxidative stress. The liver support system of claim 15, wherein the one or more genetic alterations to confer resistance to oxidative stress comprise expression of a constitutively active variant of Nuclear factor erythroid 2-related factor 2 (NRF2), mouse L- gluconolactone oxidase (mGULO), mutant albumin domain 2 comprising F211R and R218L amino acid substitutions, or MSRP5. The liver support system of any one of claims 13-16, wherein the iHLCs comprise one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling. The liver support system of claim 17, wherein the one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling comprise: edits to MHC class-I and / or MHC class-II genes; suppression of expression of HLA-A, HLA-B, B2M, NLRP3, and / or CIITA; and / or induction of expression of HLA-E and / or anti-IL1b. 31 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The liver support system of claim 17 or 18, wherein the one or more genetic alterations to reduce immunogenicity and / or inflammatory signaling comprise reduced or substantially no expression of one or more protein selected from the group consisting of HLA-A, HLA-B, and CIITA. The liver support system of claim 19, wherein the iHLC has reduced or substantially no expression of each of HLA-A, HLA-B, and CIITA. The liver support system of claim 19 or 20, wherein the expression of the one or more proteins is reduced by gene editing, optionally by CRISPR-Cas9-mediated gene editing. The liver support system of any one of claims 13-21, wherein the iHLCs comprise one or more genetic alterations to improve longevity of the iHLCs. The liver support system of claim 22, wherein the one or more genetic alterations to improve longevity comprise one or more alterations to genes involved in cell cycle, apoptosis, and / or metabolism. The liver support system of claim 23, wherein the one or more alterations to genes involved in metabolism comprise alterations to genes involved in fatty acid oxidation. The liver support system of any one of claims 13-24, wherein the iHLCs comprise one or more genetic alterations to improve detoxification function and / or hepatic synthetic function of the iHLCs. The liver support system of claim 25, wherein the one or more genetic alterations to improve detoxification function and / or hepatic synthetic function comprise: one or more genetic alterations to genes involved in albumin affinity, albumin functionality, urea cycle, and / or bilirubin clearance; coagulation factor genes and / or cytochrome P450 genes; and / or suppression of expression of ACMSD, PARP, CD38, and / or CD73. 32 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The liver support system of any one of claims 13-26, wherein the iHLCs comprise one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver. The liver support system of claim 27, wherein the one or more genetic or chemical alterations to increase regeneration of a native or transplanted liver comprise ectopic expression of HNF4A, TBX3, FOXA1, FOXA2, FOXA3, PROX1, HNF1A, HNF1B, CEBPA, NR1I2, KDM2B, GATA4, HNF6, ATF5, HHEX, NANOG, SOX2, OCT3, OCT4, c-MYC, CHIR99021, RepSox, Forskolin, Valproic acid, Parnate, TTNPB, and / or DZNep. The liver support system of any one of claims 1-28, wherein the iHLCs comprise inducible expression of one or more anti-apoptotic proteins. The liver support system of claim 29, wherein the anti-apoptotic protein is a BCL2 family protein. The liver support system of claim 29, wherein the anti-apoptotic protein is selected from the group consisting of BCL2, BLC2L1 (BCL-XL), BAX, IAP1, BIRC3,XIAP, NAIP, BIRC5, BIRC7, ILP2, and BIRC6. The liver support system of any one of claims 29-31, wherein the anti-apoptotic protein is BCL2. The liver support system of any one of claims 29-31, wherein the anti-apoptotic protein is BLC2L1 (BCL-XL). The liver support system of any one of claims 13-33, wherein the iHLCs are comprised within a two-dimensional culture, a three-dimensional culture, a cellular aggregate, a tissue section, or one or more liver organoids. The liver support system of claim 34, wherein the iHLCs are comprised within one or more liver organoids. The liver support system of claim 34 or 35, wherein the iHLCs are encapsulated within alginate capsules. 33 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The liver support system of any one of claims 1-36, wherein the system comprises one or more modifications to reduce mast cell activity. The liver support system of any one of claims 1-37, wherein the system is configured for the fluid to flow at a rate of 30-600 mL / hr. The liver support system of any one of claims 1-38, wherein the system comprises a peristaltic pump. The liver support system of any one of claims 1-39, wherein the system is configured to allow to fluid to flow (1) from a subject, (2) to the apheresis column, (3) to the detoxification column, and (4) back to the subject. A method of treating a liver disease in a subject in need thereof, the method comprising flowing whole blood from the subject through the liver support system of any one of claims 1-40. The method of claim 41, wherein the subject is afflicted with is acute-on-chronic liver failure (ACLF), acute liver failure (ALF), liver failure due to genetic inborn errors of metabolism, liver failure due to mitochondrial disease, acute fatty liver of pregnancy, intractable pruritus, drug-induced liver injury (DILI), ischemic hepatitis, cholestatic liver disease, or liver failure or liver injury following liver mass resection. The method of claim 42, wherein the liver disease is ACLF. The method of claim 43, wherein the ACLF is ACLF-1, ACLF-2, or ACLF-3. The method of claim 43 or 44, wherein the subject is afflicted with viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis of any etiology, sepsis, or systemic inflammatory response syndrome. The method of any one of claims 41-45, wherein the subject is awaiting liver transplant. The method of any one of claims 41-45, wherein the subject has undergone liver transplant or liver resection. 34 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The method of any one of claims 41-47, wherein the subject is receiving or has received terlipressin, continuous renal replacement therapy, plasmapheresis, blood filtration dialysis, and / or plasma exchange. The method of any one of claims 41-48, wherein the subject’s blood is flown through the liver support system for a period of at least two hours, at least three hours, at least six hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 38 hours, at least 42 hours, or at least 48 hours. An engineered hepatocyte-like cell comprising: reduced or substantially no expression of one or more protein selected from the group consisting of HLA-A, HLA-B, and CIITA; and engineered, optionally inducible, expression of one or more anti-apoptotic proteins. The engineered cell of claim 50, wherein the anti-apoptotic protein is a BCL2 family protein. The engineered cell of claim 50, wherein the anti-apoptotic protein is selected from the group consisting of BCL2, BLC2L1 (BCL-XL), BAX, IAP1, BIRC3,XIAP, NAIP, BIRC5, BIRC7, ILP2, and BIRC6. The liver support system of any one of claims 50-52, wherein the anti-apoptotic protein is BCL2. The liver support system of any one of claims 50-52, wherein the anti-apoptotic protein is BLC2L1 (BCL-XL). The engineered cell of any one of claims 50-54, wherein expression of the one or more anti-apoptotic protein is under the control of a doxycycline-inducible promoter. The engineered cell of any one of claims 50-55, wherein the cell has reduced or substantially no expression of each of HLA-A, HLA-B, and CIITA. 35 IPTS / 128597941.1Attorney Docket No. KNZ-001WO The engineered cell of any one of claims 50-56, wherein the expression of the one or more proteins is reduced by gene editing, optionally by CRISPR-Cas9-mediated gene editing. 36 IPTS / 128597941.1

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