Engineered hepatocytes for secreting polypeptides

Abstract

The present disclosure provides engineered hepatocytes or hepatocyte progenitor cells that secrete an incretin, such as glucagon-like peptide-1 (GLP-1) receptor agonist (RA). The hepatocytes or hepatocyte progenitor cells are engineered with an exogenous polynucleotide that includes a promoter sequence; a secretory signal; and a coding sequence encoding the polypeptide. The coding sequence is operably linked to the promoter sequence and the secretory signal to drive expression and secretion of the incretin.

Description

[0001] ENGINEERED HEPATOCYTES FOR SECRETING POLYPEPTIDES

[0002] Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 28, 2025, is named “51540-043WO2_Sequence_Listing_4_28_25” and is 10,844 bytes in size.

[0004] Background of the Invention

[0005] The global prevalence of obesity is on the rise, leading to a range of other health issues. However, there is still a lack of effective, long-term, non-invasive treatments for obesity and other metabolic disorders, such as diabetes. Glucagon-like peptide-1 (GLP-1 ) is a small peptide hormone that is secreted by various cells within the body. GLP-1 is an incretin with the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing insulin secretion and has been developed for treatment of obesity and diabetes. However, effective delivery of incretins, such as GLP-1 and other related receptor agonists has remained challenging due to the prevalence of undesired side effects. Thus, improved strategies are needed for safe and efficacious incretin-based therapies.

[0006] Summary of the Invention

[0007] In one aspect, the invention features an engineered hepatocyte or hepatocyte progenitor cell that includes a polynucleotide encoding an incretin. The incretin may be, for example, a glucagon-like peptide- 1 (GLP-1 ) receptor agonist (RA), a gastric inhibitory peptide (GIP) RA, or a glucagon RA. The polynucleotide includes a promoter sequence, a secretory signal sequence, and a coding sequence encoding the GLP-1 RA linked to the promoter sequence and the secretory signal sequence.

[0008] In some embodiments, the polynucleotide is an episomal vector within the hepatocyte or hepatocyte progenitor cell.

[0009] In some embodiments, the polynucleotide is integrated into a genome of the hepatocyte or hepatocyte progenitor cell. In some embodiments, the polynucleotide is integrated into a safe harbor locus of the genome of the hepatocyte or hepatocyte progenitor cell.

[0010] In some embodiments, the GLP-1 RA is GLP-1 , GLP-2, proglucagon, oxyntomodulin, exendin-4, cholecystokinin (CCK), or a biologically active fragment thereof.

[0011] In some embodiments, the GIP RA is GIP.

[0012] In some embodiments, the glucagon RA is glucagon.

[0013] In some embodiments, the GLP-1 is GLP-1 (1 -37), GLP-1 (7-36), or GLP-1 (7-37).

[0014] In some embodiments, the incretin (e.g., GLP-1 RA) is fused to a half-life extending moiety. In some embodiments, the half-life extending moiety is a fragment crystallizable (Fc) domain, albumin, an albumin binding peptide, an albumin binding domain (e.g., a Streptococcal protein G albumin-binding domain), a lipid binding moiety, a cholesterol binding moiety, a glycosylation site (e.g., an O-linked glycosylation site or N-linked glycosylation site), or an antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the secretory signal sequence is a human oncostatin M (OSM), vesicular stomatitis virus G protein (VSV-G), mouse immunoglobulin (Ig) kappa, mouse Ig heavy, osteonectin, SPARC [secreted protein, acidic, rich in cysteine] (BM40), secrecon, human IgKVIll, mouse IgKVIll, cluster differentiation 33 (CD33), tissue plasminogen activator (tPA), human chymotrypsinogen, human trypsinogen-2, human interleukin 2 (IL-2), gaussia luciferase, human serum albumin (HSA), influenza haemagglutinin, human insulin, or silkworm fibroin light chain (LC) signal sequence.

[0016] In some embodiments, the secretory signal sequence is located at an N-terminus of the polypeptide.

[0017] In some embodiments, the promoter is an endogenous promoter.

[0018] In some embodiments, the promoter is an exogenous promoter.

[0019] In some embodiments, the exogenous promoter is a constitutive promoter or an inducible promoter.

[0020] In some embodiments, the constitutive promoter is a cytomegalovirus (CMV), a CMV early enhancer element, a first exon and a first intron of chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene (CAG), chicken beta actin (CBA), CBA hybrid (CBh) , human elongation factor-1 alpha (EF1 a), EF1 alpha short (EFS), spleen focus-forming virus (SFFV), ubiquitin C (UBC), murine stem cell virus (MSCV), simian virus 40 (SV40), phosphoglycerate kinase (PGK), minimal cytomegalovirus promoter (minCMV), minimal herpes simplex virus thymidine kinase (minTK), a U6 promoter, a y-Box (yb) promoter.

[0021] In some embodiments, the inducible promoter is a chemically inducible promoter, such as an alcohol inducible promoter, a steroid regulated promoter, a tetracycline responsive element (TRE), a cymene repressor (CymR), a cumate switch (SparQ), a lac operon (Lac) promoter (pLAC), an arabinose inducible promoter (e.g., pBAD), an Aspergillus nidulans alcA promoter, or a LexA promoter.

[0022] In some embodiments, the inducible promoter is a temperature inducible promoter, such as a Hsp70 or Hsp90 derived promoter.

[0023] In some embodiments, the inducible promoter is a light inducible promoter, such as a lightsensing protein derived promoter (e.g., YFI or FixK2 promoter).

[0024] In some embodiments, the promoter is a physiologically responsive promoter, such as a glucoseresponsive promoter. The physiologically responsive promoter may be, for example, L-pyruvate kinase (LPK) promoter, glucose transporter-2 (GLUT2) promoter, carbohydrate response element binding protein (ChREBP) promoter, insulin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, glucose- 6-phosphatase (G6Pase) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, activating transcription factor 6 (ATF6) promoter, FAS (TNFRSF6 / CD95 / APO-1 ) promoter, or insulin-like growth factor binding protein 1 (IGFBP-1 ) promoter.

[0025] In some embodiments, the inducible promoter is a synthetic transcription factor regulatable promoter.

[0026] In some embodiments, the synthetic transcription factor is a zinc finger nuclease, a transcription activator-like effector nucleases (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 nuclease.

[0027] In some embodiments, the engineered hepatocyte or hepatocyte progenitor cell further includes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of coding sequences, each coding sequence encoding a polypeptide. For example, the engineered hepatocyte or hepatocyte progenitor cell may further include second coding sequence encoding a second polypeptide. In some embodiments, the polynucleotide encodes a linker between each coding sequence. The linker may include a cleavable peptide, such as a furin site or a 2A peptide. In some embodiments, the coding sequences are separated by an internal ribosome entry site (IRES) sequence.

[0028] In some embodiments, the second polypeptide is a dipeptidyl peptidase IV (DPP-IV) inhibitor or an incretin (e.g., a GLP-1 RA).

[0029] In some embodiments, the polynucleotide is polycistronic, e.g., bicistronic, e.g., encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes).

[0030] In some embodiments, the hepatocyte or hepatocyte progenitor cell is a hypoimmunogenic hepatocyte or hepatocyte progenitor cell. In some embodiments, the hypoimmunogenic hepatocyte or hepatocyte progenitor cell lacks expression of one or more of the following endogenous genes: beta-2- microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or a combination thereof.

[0031] In some embodiments, the hypoimmunogenic hepatocyte or hepatocyte progenitor cell includes one or more alterations that inactivate the endogenous B2M, HLA-A, HLA-B, HLA-C, CIITA, or PVR gene.

[0032] In some embodiments, the hypoimmunogenic hepatocyte or hepatocyte progenitor cell expresses one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 2 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyteactivation gene 3 (LAG-3), adenosine A2a receptor (A2AR), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), CD64, truncated CD64 (CD64t), or a combination thereof.

[0033] In some embodiments, the expression of the one or more genes is controlled by one or more inducible promoters or constitutive promoters.

[0034] In some embodiments, the hypoimmunogenic cell overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or a combination thereof relative to a wild-type hepatocyte or hepatocyte progenitor cell.

[0035] In some embodiments, the hepatocyte or hepatocyte progenitor cell is a pluripotent stem cell, such as an embryonic stem cell, an inducible pluripotent (iPSC) derived hepatocyte or hepatocyte progenitor cell or an expanded hepatocyte or hepatocyte progenitor cell. In some embodiments, the hepatocyte is a transdifferentiated hepatocyte. In some embodiments, the hepatocyte is an expanded human hepatocyte. In some embodiments, the hepatocyte is an expanded and partially reprogrammed hepatocyte or hepatocyte progenitor cell.

[0036] In another aspect, the invention features an engineered tissue construct that includes a population of hepatocytes or hepatocyte progenitor cells as described herein, e.g., of any of the above embodiments, and a biocompatible scaffold. In some embodiments, the engineered tissue construct further includes a second population of hepatocytes or hepatocyte progenitor cells, wherein the second population of hepatocytes or hepatocyte progenitor cells includes a polynucleotide encoding a second polypeptide, wherein the polynucleotide includes a promoter sequence; a secretory signal sequence; and a coding sequence encoding the second polypeptide operably linked to the promoter sequence and the secretory signal sequence.

[0037] In some embodiments, the second polypeptide is a dipeptidyl peptidase IV (DPP-IV) inhibitor or an incretin (e.g., a GLP-1 RA).

[0038] In some embodiments, the polynucleotide encoding the second polypeptide is integrated in a genome of the second population of hepatocytes or hepatocyte progenitor cells.

[0039] In some embodiments, the polynucleotide encoding the second polypeptide is integrated into a safe harbor locus of the genome of the second population of hepatocytes or hepatocyte progenitor cells.

[0040] In some embodiments, the biocompatible scaffold includes fibrin.

[0041] In some embodiments, the fibrin includes human fibrin.

[0042] In some embodiments, the biocompatible scaffold includes an immune-isolating material. In some embodiments, the biocompatible scaffold includes alginate, polyethylene glycol (PEG), modified PEG, agarose, chitosan, poly(lactic-co-glycolic acid) (PLGA), cellulose acetate, or a combination thereof.

[0043] In some embodiments, the engineered tissue construct further includes a population of stromal cells or stromal-like cells (e.g., mesenchymal progenitor cells, hepatic stellate cells, mesenchymal stem cells, or pluripotent stem cell derived mesenchymal stem cells). In some embodiments, the stromal cells include fibroblasts. In some embodiments, the fibroblasts are normal human dermal fibroblasts. In some embodiments, the stromal or stromal-like cells (e.g., fibroblasts) are hypoimmunogenic. In some embodiments, the stromal or stromal-like cells (e.g., fibroblasts) are engineered to secrete an incretin (e.g., a GLP-1 RA).

[0044] In another aspect, the invention features a method of expressing an incretin (e.g., GLP-1 RA) in a human subject. The method includes implanting an engineered hepatocyte or hepatocyte progenitor cell or an engineered tissue construct as described herein, e.g., of any of the above embodiments, thereby expressing the incretin in the human subject. In some embodiments, the method treats a disease or disorder in the subject. In some embodiments, the disease or disorder is diabetes (e.g., type 2 diabetes) or obesity.

[0045] In some embodiments, the method treats a cardiovascular disease (e.g., high blood pressure (hypertension), arrhythmia, valve disease, coronary artery disease, heart failure, peripheral artery disease, aortic disease, pericardial disease, cerebrovascular disease, deep vein thrombosis (DVT), or a congenital heart disease), a neurodegenerative disease (e.g., dementia, Alzheimer’s disease, frontotemporal dementia, chronic traumatic encephalopathy, Lewy body dementia, multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), or prion disease), a metabolic dysfunction- associated steatohepatitis (e.g., non-alcoholic steatohepatitis (NASH), or another liver condition).

[0046] In another aspect, the invention features a method of producing the engineered hepatocyte or hepatocyte progenitor cell of any of the above embodiments by packaging the polynucleotide in a viral vector and transducing the hepatocyte or hepatocyte progenitor cell with the viral vector.

[0047] In some embodiments, the viral vector is a lentiviral vector. In another aspect, the invention features a method of producing the engineered hepatocyte or hepatocyte progenitor cell of any of the above embodiments by contacting the hepatocyte or hepatocyte progenitor cell with the polynucleotide, e.g., by inserting the polynucleotide into a locus of the hepatocyte or hepatocyte progenitor cell.

[0048] In some embodiments, the method further includes introducing one or more single guide RNAs (sgRNA) into the hepatocyte or hepatocyte progenitor cell that specifically hybridizes to the locus.

[0049] In some embodiments, the method further includes providing a nuclease (e.g., a Cas9 nuclease) that introduces a double stranded DNA break to create the insertion site.

[0050] In some embodiments, the method includes introducing the polynucleotide by viral transduction, transformation, or other suitable approaches (e.g., targeted integration, which may include use of a sitespecific nuclease, transposase, transcription activator-like effector nuclease (TALEN), meganuclease, zinc finger nuclease, or a CRISPR / Cas9-based approach, homologous recombination, base editing, prime editing, transposon mediated delivery, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, diethylaminoethyl (DEAE)-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, and nanoparticle-mediated nucleic acid delivery, e.g., lipid-nanoparticle mediated nucleic acid delivery).

[0051] Definitions

[0052] As used herein, the term “about” refers to a value that is ± 10% of a reference value.

[0053] The term “adherence material” is a material incorporated into a cell mixture disclosed herein to which a cell or microorganism has some affinity, such as a binding agent. The material can be incorporated, for example, into a hydrogel. The material and a cell or microorganism interact through any means including, for example, electrostatic or hydrophobic interactions, covalent binding, or ionic attachment. The material may include, but is not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cellular receptors.

[0054] As used herein, the term “administering,” “administration,” and the like refer to giving a dosage of a composition (e.g., a cell or cell mixture, an engineered tissue construct, or an immunosuppressive agent) to a subject. The compositions utilized in the methods described herein can be administered, for example, parenterally, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by catheter, or in lipid compositions. The administration may be local or systemic. The route of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0055] As used herein, a scaffold (e.g., a hydrogel scaffold) is considered “biocompatible” when it does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of the present disclosure, it is preferable that the biocompatible scaffold does not exhibit toxicity towards the cells of the engineered tissue construct or when implanted in vivo in a subject (e.g., a human). For example, with respect to hepatocytes, hepatotoxicity can be measured, for example, by determining hepatocytes apoptotic death rate (e.g., wherein an increase in apoptosis is indicative of hepatotoxicity), transaminase levels (e.g., wherein an increase in transaminase levels is indicative of hepatotoxicity), ballooning of the hepatocytes (e.g., wherein an increase in ballooning is indicative of hepatotoxicity), microvesicular steatosis in the hepatocytes (e.g., wherein an increase in steatosis is indicative of hepatotoxicity), biliary cells death rate (e.g., wherein an increase in biliary cells death rate is indicative of hepatotoxicity), y-glutamyl transpeptidase (GGT) levels (e.g., wherein an increase in GGT levels is indicative of hepatotoxicity). A biocompatible scaffold can include, but is not limited to, fibrin and heparin. The biocompatible scaffold may be a biocompatible hydrogel scaffold.

[0056] Cells can be from established cell lines, or they can be primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from and allowed to grow in vitro for a limited number of passages, e.g., splitting, of the culture. For example, primary cultures can be cultures that have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Primary cell lines can be maintained for fewer than 10 passages in vitro. If the cells are primary cells, such cells can be harvested from an individual by any convenient method. For example, cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most conveniently harvested by biopsy. An appropriate solution can be used for dispersion or suspension of the harvested cells. Such solution will generally be a balanced salt solution, e.g., normal saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The cells can be used immediately, or they can be stored, frozen for long periods of time, being thawed, and capable of being reused. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells. For example, hepatocytes may be isolated by conventional methods which can be adapted for human liver biopsy or autopsy material (e.g., to garner primary human hepatocytes). Primary cells may be expanded and / or matured in vitro prior to administration or implantation into a subject.

[0057] As used herein, the term “cell type” refers to a group of cells sharing a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those that are isolated from a common tissue (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those that are isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0058] As used herein, an “engineered” cell (e.g., engineered hepatocyte) is a hepatocyte that includes at least one synthetic genetic modification.

[0059] As used herein, an “engineered tissue construct” refers to a mixture of cultured cells (e.g., hepatocytes), and a biocompatible scaffold (e.g., a biocompatible hydrogel scaffold, e.g., fibrin). The engineered tissue construct may further include a second population of cultured cells (e.g., fibroblasts, e.g., normal human dermal fibroblasts).

[0060] As used herein, the term “episomal” refers to the extra-chromosomal state of a plasmid in a cell. Episomal vectors (e.g., episomal plasmids) are polynucleotide molecules that are not part of the chromosomal DNA and replicate independently thereof. In one embodiment, the episomal vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In a particular aspect, the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.

[0061] As used herein, the term “hydrogel” refers to a network of polymer chains that are hydrophilic in nature, such that the material absorbs a high volume of water or other aqueous solution. Hydrogels can include, for example, at least 70% volume / volume (v / v) water, at least 80% v / v water, at least 90% v / v water, at least 95%, 96%, 97%, 98% and even 99% or greater v / v water (or other aqueous solution). Hydrogels can include natural or synthetic polymers, the polymeric network often featuring a high degree of crosslinking. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. Hydrogels are particularly useful in tissue engineering applications as scaffolds for culturing cells. In certain embodiments, the hydrogels are made of biocompatible polymers.

[0062] As used herein, the term “hypoimmunogenic cell” or “hypoimmune cell” refers to a cell that gives rise to a reduced immunological rejection response when transferred into an allogeneic host, e.g., compared to a wild-type or immunogenic cell of the same cell type. In some embodiments, hypoimmunogenic cells do not give rise to an immune response. Thus, “hypoimmunogenic” may refer to a significantly reduced or eliminated immune response when compared to the immune response of a cell prior to immuno-engineering. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof. In other embodiments, a hypoimmunogenic cell has reduced expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell expresses one or more of cluster of differentiation cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), CD64, truncated CD64 (CD64t), or any combination thereof. In some embodiments, a hypoimmunogenic cell overexpresses one or more of CD47, HLA-C, HLA-E, HLA- G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and expresses (e.g., overexpresses) one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL- 10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof.

[0063] As used herein, the terms “implanting,” “implantation,” and the like refer to directly placing one or more cells, cell mixtures, or engineered tissue constructs in any effective implantation site, such as a site that is suitable for neovascularization in a subject (e.g., a human subject). Exemplary implantation sites include an extraperitoneal space, an extrapleural space, a surface of the liver (e.g., on the surface of a renal capsule), a muscle site (e.g., a surface of a muscle, within a muscle sheath, or beneath a muscle, including but not limited to the following muscles: a rectus abdominis, an abdominal oblique, a transversus abdominus, a quadriceps femoris, a gluteus maximus, a semimembranosus, a semitendinosus, a biceps femoris, a deltoid, a biceps, or a latissimus dorsi), a pleural space, an omentum site (e.g., an omentum pedicle flap, an omentum free flap, an omental bursa, or the omentum in situ), a subcutaneous site, a surface of the pancreas, a surface of the spleen, a surface of the kidney, a bone marrow site, a bursa site, a peritoneal cavity site (e.g., a mesentery site), and / or a lesser sac site, among others.

[0064] As used herein, the term “level” refers to a level of a protein, as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” and an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more; a decrease or an increase of more than 10%, 15%, 20%, 50%, 75%, 100%, or 200%, as compared to a reference; a decrease or an 15 increase by less than 0.01 -fold, 0.02-fold, 0.1 -fold, 0.3-fold, 0.5-fold, 0.8-fold, or less; or an increase by more than 1 .2-fold, 1 .4-fold, 1 .5-fold, 1 .8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0- fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or more). A level of a protein may be expressed in mass / vol (e.g., g / dL, mg / mL, pg / mL, or ng / mL) or percentage relative to total protein in a sample.

[0065] As used herein, the term “non-native polypeptide” refers to a polypeptide that is not normally expressed in the cell type (e.g., hepatocyte) or is not expressed by the same organism (e.g., a human). Non-native polypeptides include non-naturally occurring polypeptides, such as engineered fusion proteins (e.g., GLP-1 fused to a half-life extending moiety). A non-native polypeptide may be a heterologous polypeptide.

[0066] As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change, such as the progression of a disease or disorder, e.g., obesity or diabetes (e.g., type 1 diabetes or type 2 diabetes). For example, for obesity, beneficial or desired clinical results may include, but are not limited to, weight loss or reduced blood pressure.

[0067] As used herein, the term “suitable for neovascularization” refers to conditions and / or environmental characteristics fit for the formation of new blood vessels. Generally, neovascularization means the formation of new blood vessels in injured tissue or in tissue not normally containing blood vessels or the formation of novel blood vessels (e.g., arterioles, venules, and capillaries) of a higher density than usual in said tissue. For example, a site that is suitable for neovascularization may have an existing microvessel density of greater than 3.6 vessels / mm2(e.g., greater than 3.7 vessels / mm2, 3.8 vessels / mm2, 3.9 vessels / mm2, 4 vessels / mm2, 4.1 vessels / mm2, 4.2 vessels / mm2, 4.3 vessels / mm2, 4.4 vessels / mm2, 4.5 vessels / mm2, 5 vessels / mm2, 6 vessels / mm2, 7 vessels / mm2, 8 vessels / mm2, 9 vessels / mm2, 10 vessels / mm2, 50 vessels / mm2, 100 vessels / mm2, 200 vessels / mm2, 300 vessels / mm2, 400 vessels / mm2, 500 vessels / mm2, 1000 vessels / mm2, 2000 vessels / mm2, 3000 vessels / mm2, 4000 vessels / mm2, or 4500 vessels / mm2).

[0068] As used herein, an “extraperitoneal space” refers to a space outside the peritoneal cavity, which is the cavity containing the organs in the abdomen. An extraperitoneal space may be, for example, a preperitoneal space, a retroperitoneal space, or a subperitoneal space. A preperitoneal space is the space between the peritoneum internally and the transversalis fascia externally. Organs in the preperitoneal space include, for example, the liver, spleen, stomach, superior part of the duodenum, jejunum, ileum, transverse colon, sigmoid colon and superior part of the rectum. A retroperitoneal space is the area in the back of the abdomen behind the peritoneum. Organs in the retroperitoneal space include, for example, kidneys, adrenal glands, pancreas, nerve roots, lymph nodes, abdominal aorta, inferior vena cava, and parts of the duodenum and colon. A subperitoneal space is a continuous interconnecting space beneath the peritoneum containing the extraperitoneal space, the ligaments and mesenteries, and their suspended organs. Organs in the subperitoneal space include, for example, the bladder, the cervix of the uterus, and the last part of the rectum. An engineered tissue construct that is implanted in an extraperitoneal space (e.g., preperitoneal space, a retroperitoneal space, or a subperitoneal space) is implanted on a surface within the extraperitoneal space.

[0069] As used herein, an “extrapleural space” refers to a region between the inner surface of the ribs and intercostal muscles on one side and the parietal pleura on the other. An engineered tissue construct that is implanted in an extrapleural space is implanted on a surface within the extrapleural space.

[0070] As used herein, a “pleural space” or “pleural cavity” is a cavity that exists between the parietal and visceral pleura. An engineered tissue construct that is implanted in a pleural space is implanted on a surface within the pleural space.

[0071] As used herein, an “omentum” is a large flat adipose tissue layer nestling on the surface of the intra-peritoneal organs. The greater omentum is a large apron-like fold of visceral peritoneum that hangs down from the stomach. The lesser omentum is the double layer of peritoneum that extends from the liver too the lesser curvature of the stomach, and to the first part of the duodenum.

[0072] As used herein, a “lesser sac” or “omental bursa” refers to the cavity in the abdomen that is formed by the lesser and greater omentum.

[0073] As used herein, the term “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, deer, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human.

[0074] Brief Description of the Drawings

[0075] FIG. 1 is schematic drawing showing the cell engineering workflow timeline for expansion, transduction, antibiotic selection, and detection of GLP-1 .

[0076] FIG. 2 is a schematic drawing showing the construct design for GLP-1 (7-37). The construct includes a human Ig kappa light chain V-lll region signal peptide sequence, a furin protease recognition site, and GLP-1 (7-37). The construct further included an SFFV constitutive promoter and a puromycin selection marker.

[0077] FIG. 3 is a graph quantifying DPP-4 RNA in various cells types.

[0078] FIG. 4 is a graph showing GLP-1 secretion a detected by ELISA.

[0079] FIG. 5 is a schematic drawing showing a GLP-1 activity assay workflow.

[0080] Detailed Description

[0081] The present invention features hepatocytes and hepatocyte progenitor cells that are engineered to secrete an incretin, such as a glucagon-like peptide-1 (GLP-1 ) receptor agonist (RA). The present disclosure is based, in part, on the observation that hepatocytes or hepatocyte progenitor cells can be engineered with an exogenous polynucleotide to secrete polypeptides, such as an incretin (e.g., a GLP-1 RA). In general, the invention features engineered hepatocytes or hepatocyte progenitor cells and engineered tissues constructs containing the same that include an exogenous polynucleotide encoding an incretin, such as a GLP-1 RA. The polynucleotide includes (a) a promoter sequence; (b) a secretory signal; and (c) a coding sequence encoding the incretin (e.g., GLP-1 RA) operably linked to the promoter sequence and the secretory signal. In some embodiments, the polynucleotide is integrated into a genome of the hepatocyte. In some embodiments, the polynucleotide is an episomal vector within the hepatocyte. By including a secretory signal, the polypeptides can be efficiently secreted in the extracellular milieu, where the polypeptide can impart a therapeutic function.

[0082] The exogenous polynucleotides described herein may be integrated into a genome of the hepatocyte. By integrating the polynucleotide into the genome, the cells can stably secrete the polypeptides encoded by the coding sequence without risk of losing, for example, a standard plasmid encoding the polypeptide that may easily degrade over time or get lost during cell division. In some embodiments, the polynucleotide is integrated into a safe harbor locus of the genome of the hepatocyte. Such integration can be achieved by transducing the cell with a viral vector, such as a lentiviral vector.

[0083] Engineered hepatocytes or hepatocyte progenitor cells can be aggregated with a population of stromal cells (e.g., fibroblasts) or other hepatocytes (e.g., also engineered to secrete a desired polypeptide). Such aggregates can be incorporated into an engineered tissue construct that includes a population of the engineered hepatocytes and a biocompatible scaffold. These engineered tissue constructs may further include a second population of cells (e.g., stromal cells or stromal-like cells, such as fibroblasts, or parenchymal cells, mesenchymal progenitor cells, mesenchymal stem cells, pluripotent stem cell derived mesenchymal stem cells, hepatic stellate cells, hepatocytes, e.g., primary human hepatocytes). For example, the engineered tissue constructs may include a second population of engineered hepatocytes that are engineered to secrete a different polypeptide. The engineered tissue construct may further include a population of fibroblasts. In some embodiments, the stromal or stromal- like cells (e.g., fibroblasts) are hypoimmunogenic. In some embodiments, the stromal or stromal-like cells (e.g., fibroblasts) are engineered to secrete an incretin (e.g., a GLP-1 RA).

[0084] Also featured are methods of making the engineered hepatocytes and hepatocyte progenitor cells and engineered tissue constructs and methods of use thereof, e.g., for treating a disease, such as obesity or diabetes.

[0085] Exogenous Polynucleotides

[0086] The hepatocytes described herein include one or more exogenous polynucleotides. The hepatocytes described herein may be engineered by incorporating a polynucleotide as described herein into a hepatocyte, e.g., by viral transduction, transformation, or other suitable approaches (e.g., targeted integration, which may include use of a site-specific nuclease, transposase, or a CRISPR / Cas9-based approach). For example, the hepatocyte may be transduced with the exogenous polynucleotide. The polynucleotides described herein include a promoter sequence; a secretory signal; and a coding sequence encoding a polypeptide. The coding sequence is operably linked to the promoter sequence and the secretory signal to allow for efficient expression and secretion of the polypeptide.

[0087] In some embodiments, the exogenous polynucleotide is an episomal vector within the hepatocyte. Episomal vectors (e.g., episomal plasmids) are polynucleotides that are not part of the genome of the cell and replicate independent of the eukaryotic chromosome. However, episomal vectors may still replicate together with the rest of the genome and subsequently associated with metaphase chromosomes during mitosis. Advantageously, episomes do not degrade.

[0088] Any suitable coding sequence may be included in the polynucleotides described herein. In some embodiments, the polypeptide is an incretin, e.g., a GLP-1 RA, e.g., a polypeptide of Table 1 .

[0089] Any suitable signal sequence can be used known to one of skill in the art. The signal sequence can be located at an N-terminal or C-terminal portion of the polypeptide. In some embodiments, the secretory signal sequence is located at an N-terminus of the polypeptide. In some embodiments, the secretory signal sequence is a human oncostatin M (OSM), vesicular stomatitis virus G protein (VSV-G), mouse immunoglobulin (Ig) kappa, mouse Ig heavy, osteonectin, SPARC [secreted protein, acidic, rich in cysteine] (BM40), secrecon, human IgKVIll, mouse IgKVIll, cluster differentiation 33 (CD33), tissue plasminogen activator (tPA), human chymotrypsinogen, human trypsinogen-2, human interleukin 2 (IL-2), gaussia luciferase, human serum albumin (HSA), influenza haemagglutinin, human insulin, or silkworm fibroin light chain (LC) signal sequence.

[0090] Any suitable promoter known to one of skill in the art can be used that is sufficient to drive expression of the coding sequence in the particular cell type (e.g., hepatocyte or fibroblast). In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the exogenous promoter is a constitutive promoter or an inducible promoter.

[0091] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a cytomegalovirus (CMV), a CMV early enhancer element, a first exon and a first intron of chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene (CAG), chicken beta actin (CBA), CBA hybrid (CBh) , human elongation factor-1 alpha (EF1 a), EF1 alpha short (EFS), spleen focus-forming virus (SFFV), ubiquitin C (UBC), murine stem cell virus (MSCV), simian virus 40 (SV40), phosphoglycerate kinase (PGK), minimal cytomegalovirus promoter (minCMV), minimal herpes simplex virus thymidine kinase (minTK), a U6 promoter, or y-Box (yb) promoter.

[0092] In some embodiments, the inducible promoter is a chemically inducible promoter, such as an alcohol inducible promoter, a steroid regulated promoter, a tetracycline responsive element (TRE), a cymene repressor (CymR), a cumate switch (SparQ), a lac operon (Lac) promoter (pLAC), an arabinose inducible promoter (e.g., pBAD), an Aspergillus nidulans alcA promoter, or a LexA promoter.

[0093] In some embodiments, the inducible promoter is a temperature inducible promoter, such as a Hsp70 or Hsp90 derived promoter.

[0094] In some embodiments, the inducible promoter is a light inducible promoter, such as a lightsensing protein derived promoter (e.g., YFI or FixK2 promoter).

[0095] In some embodiments, the promoter is a physiologically responsive promoter, such as a glucoseresponsive promoter. The physiologically responsive promoter may be, for example, L-pyruvate kinase (LPK) promoter, glucose transporter-2 (GLUT2) promoter, carbohydrate response element binding protein (ChREBP) promoter, insulin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, glucose- 6-phosphatase (G6Pase) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, activating transcription factor 6 (ATF6) promoter, FAS (TNFRSF6 / CD95 / APO-1 ) promoter, or insulin-like growth factor binding protein 1 (IGFBP-1 ) promoter.

[0096] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive element (TRE), cymene repressor (CymR), cumate switch (SparQ), or lac operon (Lac) promoter.

[0097] In some embodiments, the inducible promoter is a synthetic transcription factor regulatable promoter. In some embodiments, the synthetic transcription factor is a zinc finger nuclease, a transcription activator-like effector nucleases (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 nuclease.

[0098] In some embodiments, the polynucleotide may include other regulatory elements, such as enhancers, one or more IRES sequences, and the like.

[0099] In some embodiments, the engineered hepatocytes described herein further include a second coding sequence encoding a second polypeptide. The second coding sequence may be present on the same exogenous polynucleotide, such that the polynucleotide encodes two polypeptides, e.g., in a polycistronic (e.g., bicistronic) vector, e.g., encodes 2, 3, 4, 5, 6 , 7, 8, 9, 10, or more genes). In some embodiments, the second coding sequence is present on a second exogenous polynucleotide. In some embodiments, the second exogenous polynucleotide is integrated into another location (e.g., safe harbor locus) of the genome of the hepatocyte that is different from the first location. In some embodiments, the second exogenous polynucleotide is present within a second episomal vector.

[0100] In some embodiments, the polynucleotide encodes a linker between each coding sequence. The linker may include a cleavable peptide, such as a furin site or a 2A peptide. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is a foot-and-mouth disease virus 18 2A (F2A) peptide, an equine rhinitis A virus 2A (E2A) peptide, a porcine teschovirus-1 2A (P2A) peptide, or a thosea asigna virus 2A (T2A) peptide. In some embodiments, the polynucleotide encodes a 2A peptide in between the first and second coding sequences, optionally with a flexible linker flanking the 2A peptide (e.g., GSG linker). The coding sequence may further include one or more engineered cleavage sequences, e.g., a furin cleavage sequence to remove the 2A peptide residues attached to the peptide. Exemplary 2A peptides are described, e.g., in Chang et al MAbs 7: 403- 412 (2015) and Lin et al. Front. Plant Sci. 9:1379 (2018), the disclosures of which are hereby incorporated by reference in their entirety.

[0101] In some embodiments, the coding sequences are separated by an internal ribosome entry site (IRES) sequence.

[0102] In some embodiments, the second polypeptide is a dipeptidyl peptidase IV (DPP-IV) inhibitor. In some embodiments, the second polypeptide is an incretin, e.g., a GLP-1 RA, e.g., the same or different incretin or GLP-1 RA as the first polypeptide.

[0103] In some embodiments, the exogenous polynucleotides are packaged in a viral vector (e.g., a lentiviral vector).

[0104] Incretins

[0105] The engineered hepatocytes described herein are programmed to secrete an incretin. The incretin may be, for example, a GLP-1 RA polypeptide, a gastric inhibitory peptide (GIP) RA, or a glucagon RA. The polynucleotide includes a promoter sequence; a secretory signal sequence; and a coding sequence encoding the incretin linked to the promoter sequence and the secretory signal sequence.

[0106] In some embodiments, the GLP-1 RA is GLP-1 , GLP-2, proglucagon, oxyntomodulin, exendin-4, cholecystokinin (CCK), or a biologically active fragment thereof.

[0107] In some embodiments, the GLP-1 RA is GLP-1 or a biologically active fragment thereof.

[0108] In some embodiments, the GLP-1 RA is GLP-2 or a biologically active fragment thereof.

[0109] In some embodiments, the GLP-1 RA is oxyntomodulin (OXM) or a biologically active fragment thereof.

[0110] In some embodiments, the GLP-1 RA is exendin-4 or a biologically active fragment thereof.

[0111] In some embodiments, the GLP-1 RA is CCK or a biologically active fragment thereof.

[0112] In some embodiments, the GLP-1 is GLP-1 (1 -37), GLP-1 (7-36), or GLP-1 (7-37). In some embodiments, the GLP-1 is GLP-1 (1 -37). In some embodiments, the GLP-1 is GLP-1 (7-36). In some embodiments, the GLP-1 is GLP-1 (7-37).

[0113] In some embodiments, the GIP RA is GIP or a biologically active fragment thereof.

[0114] In some embodiments, the glucagon RA is glucagon.

[0115] Exemplary incretins are shown in Table 1 below:

[0116] Table 1. Incretins

[0117] In some embodiments, the incretin is an incretin of Table 1 . In some embodiments, the incretin has the amino acid sequence of any one of SEQ ID NOs: 1 -11 .

[0118] In some embodiments, the incretin is fused to a half-life extending moiety. In some embodiments, the half-life extending moiety is a fragment crystallizable (Fc) domain, albumin, an albumin binding peptide, an albumin binding domain (e.g., a Streptococcal protein G albumin-binding domain), a lipid binding moiety, a cholesterol binding moiety, a glycosylation site (e.g., an O-linked glycosylation site or N- linked glycosylation site), or an antibody or antigen-binding fragment thereof. In some embodiments, the half-life extending moiety is an Fc domain. In some embodiments, the half-life extending moiety is albumin. In some embodiments, the half-life extending moiety is an albumin binding peptide. In some embodiments, the half-life extending moiety is an antibody or antigen-binding fragment thereof.

[0119] Methods for Making Engineered Hepatocytes

[0120] Also featured herein is a method of producing an engineered hepatocyte or hepatocyte progenitor cell as described herein. In general, the methods include incorporating a polynucleotide as described herein into a hepatocyte, e.g., by viral transduction, transformation, or other suitable approaches (e.g., targeted integration, which may include use of a site-specific nuclease, transposase, transcription activator-like effector nuclease (TALEN), meganuclease, zinc finger nuclease, a CRISPR / Cas9-based approach, homologous recombination, base editing, prime editing, transposon mediated delivery, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, diethylaminoethyl (DEAE)-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, and nanoparticle-mediated nucleic acid delivery, e.g., lipid-nanoparticle mediated nucleic acid delivery). The method may include packaging an exogenous polynucleotide with a coding sequence that encodes the polypeptide in a viral vector and transducing the hepatocyte with the viral vector. The exogenous polynucleotides described herein may then be integrated into a genome of the hepatocyte. In other embodiments, the method may include transforming the hepatocyte with an episomal vector that includes the exogenous polynucleotide.

[0121] By integrating the polynucleotide into the genome or, the cells can stably secrete the polypeptides encoded by the coding sequence without risk of losing, for example, a standard plasmid encoding the polypeptide that may easily degrade over time or get lost during cell division. In some embodiments, the polynucleotide is integrated into a safe harbor locus of the genome of the hepatocyte. Such integration can be achieved by transducing the cell with a viral vector, such as a lentiviral vector.

[0122] Any suitable safe harbor locus may be used according to one of skill in the art. For example, the safe harbor locus may be the AAVS1 locus in intron 1 of PPP1 R12C, hROSA26, CCR5, SHS231 , or SHS229. Safe harbor loci are described, e.g., in Pellenz et al. Hum. Gene Then 30:814-828, 2019, which is hereby incorporated by reference in its entirety.

[0123] In some embodiments, the hepatocyte or hepatocyte progenitor cell is a pluripotent stem cell, such as an embryonic stem cell or an inducible pluripotent (iPSC) derived hepatocyte or hepatocyte progenitor cell, or an expanded hepatocyte or hepatocyte progenitor cell. In some embodiments, the hepatocyte is a transdifferentiated hepatocyte. In some embodiments, the hepatocyte is an expanded human hepatocyte. In some embodiments, the hepatocyte is an expanded and partially reprogrammed hepatocyte or hepatocyte progenitor cell.

[0124] Viral Vectors for Expression

[0125] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous polynucleotides into a mammalian cell, e.g., a hepatocyte. The polynucleotide to be delivered may include a promoter, a coding sequence, and secretion signal. Viral genomes are particularly useful vectors for polynucleotide delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., an adeno- associated viral (AAV) vector), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T- cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (US 5,801 ,030), the teachings of which are incorporated herein by reference.

[0126] Retroviral Vectors

[0127] The delivery vector used in the methods described herein may be a retroviral vector. One type of retroviral vector that may be used in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene encoding the polypeptide or RNA. An overview of optimization strategies for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

[0128] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the agent of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1 ) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of a heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, encapsidation, and expression, in which the sequences to be expressed are inserted.

[0129] A LV used in the methods and compositions described herein may include one or more of a 5'- Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 -alpha promoter and 3'-self inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE. The lentiviral vector may further include a pHR' backbone, which may include for example as provided below.

[0130] The Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) may be used to express the DNA molecules and / or transduce cells. A LV used in the methods and compositions described herein may a 5'-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 - alpha promoter and 3'-self inactivating L TR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.

[0131] Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.

[0132] In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. The vector used in the methods and compositions described herein may include multiple promoters that permit expression of more than one polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in Klump et al., Gene Ther.; 8:811 (2001 ), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004), the disclosures of which are incorporated herein by reference as they pertain to protein cleavage sites that allow expression of more than one polypeptide. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein.

[0133] The viral vectors (e.g., retroviral vectors, e.g., lentiviral vectors) may include a promoter operably coupled to the transgene encoding the polypeptide to control expression. The promoter may be a ubiquitous promoter. Alternatively, the promoter may be a tissue specific promoter, such as a myeloid cell-specific or hepatocyte-specific promoter. Suitable promoters that may be used with the compositions described herein include CD11 b promoter, sp146 / p47 promoter, CD68 promoter, sp146 / gp9 promoter, elongation factor 1 a (EF1a) promoter, EF1a short form (EFS) promoter, phosphoglycerate kinase (PGK) promoter, a-globin promoter, and p-globin promoter. Other promoters that may be used include, e.g., DC172 promoter, human serum albumin promoter, alphal antitrypsin promoter, thyroxine binding globulin promoter. The DC172 promoter is described in Jacob, et al. Gene Ther. 15:594-603, 2008, hereby incorporated by reference in its entirety.

[0134] The viral vectors (e.g., retroviral vectors, e.g., lentiviral vectors) may include an enhancer operably coupled to the transgene encoding the polypeptide or the polynucleotide encoding the RNA to control expression. The enhancer may include a p-globin locus control region (pLCR).

[0135] Hepatocytes and Hepatocyte Progenitor Cells

[0136] The engineered cells described herein are hepatocytes or hepatocyte progenitor cells. In some embodiments, the engineered hepatocytes or hepatocyte progenitor cells are present within an engineered tissue construct. In some embodiments, the hepatocytes are primary human hepatocytes (PHH). In some embodiments, the hepatocytes or hepatocyte progenitor cells are derived from stem cells (e.g., embryonic stem cells or pluripotent stem cells). In some embodiments, the hepatocytes are adult solid organ derived hepatocytes. In some embodiments, the hepatocytes are derived from pluripotent stem cells (e.g., induced pluripotent stem cells). In some embodiments, the hepatocytes are expanded hepatocytes. In some embodiments, the hepatocyte is an expanded and partially reprogrammed hepatocyte or hepatocyte progenitor cell.

[0137] In some embodiments, the density of hepatocytes or hepatocyte progenitor cells, e.g., in an engineered tissue construct, is 0.1 M / mL to 150 M / mL (e.g., 0.2 M / mL to 149 M / mL, 0.3 M / mL to 148 M / mL, 0.4 M / mL to 147 M / mL, 0.5 M / mL to 146 M / mL, 1 M / mL to 145 M / mL, 5 M / mL to 140 M / mL, 10 M / mL to 100 M / mL, 20 M / mL to 50 M / mL, or 30 M / mL to 40 M / mL). For example, in some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 0.2 M / mL to 149 M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 0.3 M / mL to 148 M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 0.4 M / mL to 147 M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 0.5 M / mL to 146 M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 1 M / mL to 145

[0138] M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 5 M / mL to 140

[0139] M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 10 M / mL to 100

[0140] M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 20 M / mL to 50

[0141] M / mL. In some embodiments, the density of hepatocytes or hepatocyte progenitor cells is 30 M / mL to 40

[0142] M / mL.

[0143] In some embodiments, the engineered tissue construct includes a population of hepatocytes or hepatocyte progenitor cells in an amount of from 3 x 105to 1 .8 x 1011(e.g., from 4 x 105to 1 .8 x 1011, from 5 x 105to 1 .8 x 1011, from 6 x 105to 1 .8 x 1011, from 7 x 105to 1 .8 x 1011, from 8 x 105to 1 .8 x 1011, from 9 x 105to 1 .8 x 1011, from 1 x 106to 1 .8 x 1011, from 2 x 106to 1 .8 x 1011, from 3 x 106to 1 .8 x 1011, from 4 x 106to 1 .8 x 1011, from 5 x 106to 1 .8 x 1011, from 6 x 106to 1 .8 x 1011, from 7 x 106to 1 .8 x 1011, from 8 x 106to 1 .8 x 1011, from 9 x 106to 1 .8 x 1011, from 1 x 107to 1 .8 x 1011, from 2 x 107to 1 .8 x 1011, from 3 x 107to 1 .8 x 1011, from 4 x 107to 1 .8 x 1011, from 5 x 107to 1 .8 x 1011, from 6 x 107to 1 .8 x 1011, from 7 x 107to 1 .8 x 1011, from 8 x 107to 1 .8 x 1011, from 9 x 107to 1 .8 x 1011, from 1 x 108to 1 .8 x 1011, from 2 x 108to 1 .8 x 1011, from 3 x 108to 1 .8 x 1011, from 4 x 108to 1 .8 x 1011, from 5 x 108to 1 .8 x 1011, from 6 x 108to 1 .8 x 1011, from 7 x 108to 1 .8 x 1011, from 8 x 108to 1 .8 x 1011, from 9 x 108to 1 .8 x 1011, from 1 x 109to 1 .8 x 1011, from 2 x 109to 1 .8 x 1011, from 3 x 109to 1 .8 x 1011, from 4 x 109to 1 .8 x 1011, from 5 x 109to 1 .8 x 1011, from 6 x 109to 1 .8 x 1011, from 7 x 109to 1 .8 x 1011, from 8 x 109to 1 .8 x 1011, from 9 x 109to 1 .8 x 1011, from 1 x 1010to 1 .8 x 1011, from 2 x 1010to 1 .8 x 1011, from 3 x 1010to 1 .8 x 1011, from 4 x 1010to 1 .8 x 1011, from 5 x 1010to 1 .8 x 1011, from 6 x 1010to 1 .8 x 1011, from 7 x 1010to 1 .8 x 1011, from 8 x 1010to 1 .8 x 1011, from 9 x 1010to 1 .8 x 1011, or from 1 x 1011to 1 .8 x 1011) hepatocytes or hepatocyte progenitor cells.

[0144] Hypoimmunogenic Cells

[0145] Hypoimmunogenic cells are cells that give rise to a reduced or eliminated immunological rejection response when transferred into an allogeneic host. In some embodiments, the engineered hepatocytes or hepatocyte progenitor cells described herein are hypoimmunogenic hepatocytes or hepatocyte progenitor cells. In some embodiments, the stromal cells (e.g., fibroblasts) described herein are hypoimmunogenic stromal cells, e.g., fibroblasts. Hypoimmunogenic cells do not give rise to any immune response. Thus, the hypoimmunogenic cells elicit a significantly reduced or abrogated immune response when compared to the immune response of the same cell prior to immuno-engineering.

[0146] Any suitable hypoimmunogenic cells may be used in the cells, engineered tissue constructs, and methods disclosed herein. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof. In some embodiments, a hypoimmunogenic cell expresses (e.g., overexpresses) one or more of cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3- dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), CD64, truncated CD64 (CD64t), or any combination thereof. Any of the cell types described herein may be genetically modified to produce hypoimmunogenic cells. For example, hepatocytes or hepatocyte progenitor cells may be engineered to be hypoimmunogenic hepatocytes or hepatocyte progenitor cells.

[0147] In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of B2M. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-A. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-B. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-C. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of CIITA. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of PVR. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD47. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-E. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-G. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) PD-L1 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CTLA4. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses CD39. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD73. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD24. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD27. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD35. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD46. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD55. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD59. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD200. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-C. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IDO1 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IL-10. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IL-35. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) FASL. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CCL21 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) MFG-E8. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) SERPINB9. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) DUX4. In some embodiments, the hypoimmunogenic cells express a CTLA4-immunoglobulin fusion protein (CTLA4-lg). See, e.g., Rong et al. Cell Stem Cell. 14:121 -130, 2014.

[0148] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class I genes (e.g., via knockout of B2M or knockout of HLA-A, HLA-B, and / or HLA-C, e.g., knockout of HLA-A and HLA-B). In some embodiments, hypoimmunogenic cells lacking one or more HLA class I genes (e.g., via knockout of B2M or knockout of HLA-A, HLA-B, and / or HLA-C, e.g., knockout of HLA-A and HLA-B) also may express (e.g., overexpress) HLA-E (e.g., a singlechain HLA-E). See, e.g., Gornalusse et al. Nat. Biotechnol. 35:765-772, 2017.

[0149] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class II genes (e.g., via knockout of CIITA, RFXANK, RFX5, or RFXAP). In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class II genes via knockout of CIITA.

[0150] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA, or knockout of HLA-A, HLA-B, and / or HLA-C and CIITA).

[0151] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA, or knockout of HLA-A, HLA-B, and / or HLA-C and CIITA) and express (e.g., overexpress) CD47 (see, e.g., Deuse et al. Nat. Biotechnol. 37:252-258, 2019).

[0152] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA) and express (e.g., overexpress) CD47, HLA-G, and PD-L1 (see, e.g., Han et al. Proc. Natl. Acad. Sci USA 116:10441 -10446, 2019).

[0153] In some embodiments, the hypoimmunogenic cells express a mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins, e.g., to protect against allogeneic NK cell-mediated lysis (see, e.g., Guo, et al. European Journal of Immunology. 51 : 2513-2521 , 2021 ).

[0154] Additional hypoimmunogenic cells that can be used in the cells, cell mixtures, engineered tissue constructs, and methods disclosed herein include, without limitation, any hypoimmunogenic cell described in Zhao et al. iScience. 23(6):101162, 2020, WO 2018 / 132783, WO 2021 / 041316, WO 2021 / 022223, or WO 2019 / 014351. Hypoimmunogenic cells can be generated by any suitable technique known in the art to the skilled artisan. For example, cells (e.g., iPSCs or hepatocytes) can be gene edited (e.g., using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology or other gene editing technologies known in the art such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs)) to reduce expression of, knock out, express, or overexpress one or more of the genes described above. For example, one or both alleles of an endogenous gene may be knocked out to reduce or eliminate expression of the gene (e.g., B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof). In another example, a cell may be engineered to express or overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, CD64, CD64t, or any combination thereof, via transduction with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector) or an adeno-associated viral (AAV) vector)).

[0155] The expression of or more of any of the genes disclosed herein, e.g., CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, CD64, CD64t, or any combination thereof, may be controlled by any suitable control element(s), e.g., promoters or enhancers, including constitutively active promoters (e.g., the human p-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1 a, (EF1a), phosphoglycerate kinase (PGK) and ubiquitinC (UbC)) and / or inducible promoters (e.g., a tetracycline-controlled promoter or a doxycycline-inducible promoter). Other constitutively active promoters and inducible promoters are known in the art.

[0156] Methods for making engineered hypoimmunogenic cells

[0157] Methods for making engineered hypoimmunogenic cells are known in the art. Numerous approaches have been suggested to address allogeneic rejection of transplanted or engrafted cells. These include HLA-matching, blocking T-cell activation pathways using antibodies, administering a combination of immunosuppressive drugs, and employing autologous cell therapy. Another tactic to mitigate graft rejection involves minimizing the allogenic differences between the transplanted or engrafted cells and the recipient.

[0158] Human leukocyte antigens (HLAs), which are cell surface-expressed molecules encoded by genes located in the human major histocompatibility complex on chromosome 6, play a pivotal role in mediating immune rejection. A single HLA gene mismatch between the donor and recipient can trigger a robust immune response. HLA genes are categorized into MHC class I (MHC-I) and MHC class II (MHC- II). MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in nearly all tissue cell types. They present "non-self" antigen-processed peptides to CD4+ T cells, promoting their activation to cytolytic CD8+ T cells. Transplanted or engrafted cells expressing "non-self" MHC-I molecules provoke a robust cellular immune response, leading to their demise by activated cytolytic CD8+ T cells.

[0159] MHC-I proteins are closely associated with beta-2-microglobulin (B2M) in the endoplasmic reticulum and are essential for forming functional MHC-I molecules on the cell surface. Unlike the widespread cellular expression of MHC-I genes, MHC-II gene expression is restricted to antigen- presenting cells such as dendritic cells, macrophages, and B cells. HLA antigen genes exhibit the highest polymorphism observed in the human genome.

[0160] Developing a universal donor cell with stealth features, compatible with any HLA genotype, offers an alternative strategy to address immune rejection and the associated economic costs of current immune evasion methodologies.

[0161] To create a line of universal donor cells, one approach involves functionally disrupting the expression of MHC-I and MHC-II class genes. This disruption could be achieved through genetic methods, such as targeting both genetic alleles encoding the MHC-I light chain, B2M. The resulting B2M- null cell line and its derivatives may have significantly reduced surface MHC-I, resulting in diminished immunogenicity to allogeneic CD8+ T cells. The transcription activator-like effector nuclease (TALEN) targeting approach can be utilized to generate B2M-deficient stem cell lines by deleting a few nucleotides in exon 2 of the B2M gene (see, e.g., Lu, P. et al., Stem Cell Rev. 9:806-813, 2013, incorporated by reference herein). Additionally, another method achieves stem cells that evade allogeneic recognition by knocking out one B2M allele and inserting an HLA-E gene at a second B2M allele. This leads to the surface expression of HLA-E dimers or trimers without the expression of HLA-A, HLA-B, or HLA-C (see, e.g., Gornalusse, G.G. et al., Nature Biotechnology. 35(8): 765-773, 2017, incorporated by reference herein).

[0162] MHC class l-negative cells may be susceptible to lysis by natural killer (NK) cells, as HLA molecules act as major ligand inhibitors to NK cells. Host NK cells have been demonstrated to eliminate transplanted or engrafted B2M- / - donor cells, a phenomenon also observed in vitro with MHC class-l- negative human leukemic lines (see, e.g., Bix, M. et al. Nature. 349: 329-331 , 1991 ).

[0163] Post-engraftment cell survival may be influenced by various pathways independent of allogeneic rejection, such as stress responses (e.g., hypoxia, reactive oxygen species, nutrient deprivation, and oxidative stress). In some instances of, a population of human hepatocytes genetically engineered with stealth features, is described. Another embodiment involves human hepatocytes genetically engineered with both stealth features and cell survival factors to facilitate cell engraftment post-transplantation.

[0164] In certain embodiments, a polynucleotide encoding one or more tolerogenic factors can be introduced into cells, either genetically modified or unmodified, to create immune-privileged expanded human hepatocytes. Exemplary tolerogenic factors include, among others, TNFAIP3, CD39, PD-L1 , HLA- E, CD73, HLA-C, HLA-F, HLA-G, CTLA-4-lg, and CD47. The nucleotide sequence encoding a tolerogenic factor may be operably linked to an exogenous promoter, selected from constitutive, inducible, temporal-, tissue-, or cell type-specific promoters. Various eukaryotic promoters, functional in eukaryotic cells, can be utilized, including those from CMV, HSY, SV40, LTRs from retroviruses, EFla, CBA, UBC, CAG, CAGGS, MSCV, PGK, and mouse metallothionein-l. Promoters may be inducible (e.g., heat shock, tetracycline-regulated), constitutive (e.g., CMV, UBC, CAG), or spatially / temporally restricted (e.g., tissuespecific, cell type-specific).

[0165] The introduction of complexes, polypeptides, and polynucleotides into cells can occur through various methods such as viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)- mediated transfection, diethylaminoethyl (DEAE)-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, and nanoparticle-mediated nucleic acid delivery, e.g., lipid-nanoparticle mediated nucleic acid delivery.

[0166] In some embodiments, a polynucleotide encoding at least one tolerogenic factor and / or survival factor may include a sequence encoding one or more ribosome skips. This results in the production of a single transcript but, due to a ribosome skip during translation, leads to the production of two or more separate proteins. The ribosome skip can be a short peptide (around 20 amino acids) preventing the ribosome from creating the peptide bond between a glycine and a proline at the C-terminal end of the growing polypeptide chain. This mechanism results in apparent co-translational cleavage of the polypeptide. The ribosome skip peptide may belong to the 2A sequence family, with examples including F2A, T2A, E2A, and P2A, with P2A specifically mentioned in certain embodiments. Alternatively, the ribosome skip can be an internal ribosome entry sequence (IRES), allowing for translation initiation in a cap-independent manner, derived from viral or cellular sources.

[0167] In one example, support cell populations (dermal fibroblasts) of human origin may be genetically engineered using lentiviral vectors to overexpress immune checkpoint proteins such as PD-L1 , CD47, CD39 / 73 and then co-encapsulated with primary human hepatocytes (see, e.g., U.S. Pub. No. 2020 / 0164105, which is herein incorporated by reference). When co-encapsulated with parenchymal cells, the engineered supporting cell population provided inhibitory signals to nearby immune cells, thus protecting the co-encapsulated cells from T cell cytotoxicity. Inhibitors of various immune effectors may be employed, such as PD-L1 , CD47, and CD39 / 73. These four molecules can function in concert to inhibit the innate immune response, antigen presentation by dendritic cells, the adaptive immune response, and any residual immune activity. CD47 can inhibit macrophage and NK cell activity and downregulate dendritic cell activa-tion. PD-L1 is a potent T cell suppressant, while CD39 and CD73 can also be used to inhibit any residual T cell-mediated cytotoxicity that will lead to an increase in extracellular ATP concentra-tion resulting from cellular lysis. Hydrolysis of extracellular ATP by membrane-bound ectonucleotidases (CD39 and CD73) generates immunosuppressive adenosine, thus acting as a negative-feedback mechanism to prevent excessive inflammation and graft damage.

[0168] In another example, hypoimmune hepatocytes can be generated by using Cas9 protein or equivalent nuclease, selected gRNA targeting B2M or CIITA, resulting in a ribonucleoprotein (RNP) complex that can be electroplated into eHHs. Cells are washed and then suspended in an electroporation buffer, followed by the addition of two RNP complexes (one for each of the two sgRNAs) and electroporated. After electroporation, cells are diluted with media without serum, and rAAV vector or plasmid is added at the appropriate MOI.

[0169] Plasmid design to insert a transgene encoding HLA-E-P2A -PD-L1 into the B2M locus can be made such that the starting codon of B2M is removed after undergoing homology-directed repair (HDR) to insert the transgene, nullifying any chance of partial B2M expression. Successful HDR results in the insertion of the 3 genes of HLA-E, and PD-L-1 (CD274) into the genome. The two coding sequences can be linked by P2A peptide coding sequences to allow for the expression of the two separate proteins from a single transcript. The donor plasmid can contain a CAGGS promoter (i.e., comprising a CMV enhancer, a chicken 3-actin promoter, and a chimeric intron) driven cDNA of HLA-E-P2A-PD-L-1 flanked by base pair homology arms with identical sequence to the B2M locus around the cut site. Several days post electroporation, the cells can be enriched for PD-L-1 expressing cells, e.g., via magnetic assisted cell sorting (MACS) using anti-mouse IgG Dyna-beads (ThermoFisher, CELLection™ Pan Mouse IgG Kit, 1 153D). These enriched cells) represented a bulk KI population that is highly PD-L-1 positive. The enriched cells can then be FACS-sorted for PD-L-1 surface expression and seeded into BIOLAMININ 521 coated plates.

[0170] Engineered Tissue Constructs

[0171] The engineered hepatocytes or hepatocyte progenitor cells described herein may be encapsulated in an engineered tissue construct. The engineered tissue constructs described herein include a population of hepatocytes or hepatocyte progenitor cells and a biocompatible scaffold. The engineered tissue construct may further include a second population of cells, such as hepatocytes or hepatocyte progenitor cells or a population of another cell type, such as stromal cells, e.g., fibroblasts.

[0172] In some embodiments, the engineered tissue construct includes two or more populations of cells (e.g., two, three, four, five, six, seven, eight, nine, ten, or more populations of cells).

[0173] The shape of the engineered tissue construct may depend on the site of implantation and / or the disease or disorder to be treated. In some embodiments, the engineered tissue construct is triangular, rectangular (e.g., square), square with rounded corners, cylindrical, or circular. In some embodiments, the engineered tissue construct has a thickness that is substantially less than its length and width. For example, the engineered tissue construct may be substantially flat, e.g., a flat rectangle, triangle, or disc shape.

[0174] The engineered tissue construct may have a length, width, and thickness that are each, independently, from 0.1 mm to 100 cm, e.g., from 0.1 mm to 1 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm), from 1 mm to 1 cm (e.g., 1 mm, 2 mm, 3 mm,

[0175] 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 1 cm), from 1 cm to 10 cm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm), or from 10 cm to 100 cm (e.g., 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, or 100 cm). In some embodiments, the thickness of the engineered tissue construct is from 0.1 mm to 1 cm, e.g., from 0.1 mm to 1 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm) or from 1 mm to 1 cm (e.g., 1 mm, 2 mm, 3 mm, 4 mm,

[0176] 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 1 cm), and the length and / or width are each, independently, from 1 cm to 10 cm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm) or from 10 cm to 100 cm (e.g., 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, or 100 cm). In some embodiments, the length and width are each, independently, at least 5-fold (e.g., at least 5-fold, at least 6-fold, 7-fold, 8- fold, 9-fold, 10-folder, 20-fold, 30-fold, 40-fold, 50-fold, or more) greater than the thickness of the engineered tissue construct.

[0177] In some embodiments, the engineered tissue construct has a surface area of 10 cm2to 2,000 cm2, e.g., from 10 cm2to 100 cm2(e.g., 10 cm2, 20 cm2, 30 cm2, 40 cm2, 50 cm2, 60 cm2, 70 cm2, 80 cm2, 90 cm2, or 100 cm2), 100 cm2to 1 ,000 cm2(e.g., 100 cm2, 200 cm2, 300 cm2, 400 cm2, 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, or 1 ,000 cm2), or 1 ,000 cm2to 2,000 cm2(e.g., 1 ,000 cm2, 1 ,100 cm2, 1 ,200 cm2, 1 ,300 cm2, 1 ,400 cm2, 1 ,500 cm2, 1 ,600 cm2, 1 ,700 cm2, 1 ,800 cm2, 1 ,900 cm2, or 2,000 cm2).

[0178] In some embodiments, the cells are present (e.g., encapsulated) on a first face of the engineered tissue construct. In some embodiment, the cells are present (e.g., encapsulated) on a first face and a second face of the engineered tissue construct. In some embodiments, the first face of the engineered tissue construct containing the encapsulated cells contacts a site of implantation.

[0179] In some embodiments, the method includes implanting a plurality of engineered tissue constructs. For example, in some embodiments, the engineered tissue construct has a thickness that is substantially less than its length and width (e.g., a substantially flat rectangle, triangle, or disc shape) and a stack of engineered tissue constructs is implanted in a site of implantation. In other examples, the members of the plurality may be implanted in different implantation sites.

[0180] In another aspect, the cells are provided in the form of an aggregate (e.g., a spheroidal aggregate). In some embodiments, the cell populations are admixed under conditions which cause the two cell populations to form aggregates. In some embodiments, the cell populations are admixed using tissue fabrication techniques. In some embodiments, two or more cell populations are co-cultured. In some embodiments, two or more cell populations are cocultured by hanging drop, microwell molding, non-adhesive surfaces, spheroid suspension culture using a spinner flask, vertical wheel bioreactor, horizontal wheel bioreactor, or a microfluidic spheroid system. Additional methods include those using acoustical waves and using positively charged surfaces on a plate.

[0181] In other aspects, the compositions provided herein can contain additional components, including but not limited to, growth factors, ligands, cytokines, drugs, and the like. In some embodiments, the cells or cell mixtures can include molecules which elicit additional microenvironmental cues such as small molecules or growth factors which stimulate or enhance proliferation and expansion of a cell population.

[0182] The properties of the cell aggregates of the present disclosure can be varied to suit a particular application. In certain embodiments, the density of the cell aggregates can be changed. In certain embodiments, cell aggregates of different diameters can be fabricated. In certain embodiments, the overall network organization of the one or more cell aggregates can be defined, for example, by the number, three-dimensional organization, alignment, diameters, density, and the like.

[0183] In certain embodiments, the engineered tissue construct can contain one or more bioactive substances. Examples of bioactive substance(s) include, but are not limited to, hormones, neurotransmitters, growth factors, hormone, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, extracellular matrix components, and adhesion molecules, ligands and peptides; such as growth hormone, parathyroid hormone bone morphogenetic protein, transforming growth factor-alpha, TGF-beta1 , TGF-beta2, stromal cell growth factor, granulocyte / macrophage colony stimulating factor, epidermal growth factor, platelet derived growth factor, insulin-like growth factor, scatter factor / hepatocyte growth factor, fibrin, dextran, matrix metalloproteinases, collagen, fibronectin, vitronectin, hyaluronic acid, an RGD-containing peptide or polypeptide, an angiopoietin and vascular endothelial cell growth factor.

[0184] In certain embodiments, the engineered tissue constructs disclosed herein include one or more adherence materials to facilitate maintenance of the desired phenotype of the grafted cells in vivo. The material may include, for example, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cellular receptors.

[0185] The type of adherence materials (e.g., extra-cellular matrix (ECM) materials, sugars, proteoglycans etc.) will be determined, in part, by the cell type or types (e.g., hepatocytes and fibroblasts) to be cultured. ECM molecules found in a cell's native microenvironment are useful in maintaining the function of both primary cells, precursor cells, and / or cell lines.

[0186] In some embodiments, the engineered tissue construct further includes a biocompatible scaffold (e.g., biocompatible hydrogel scaffold). For example, in some embodiments, the biocompatible scaffold includes fibrin (e.g., human fibrin or human fibrinogen, e.g., FIBRYGA®). In some environments, the biocompatible scaffold is resorbable. In some embodiments, the fibrin includes polymerized fibrinogen. In some embodiments, the fibrinogen is reconstituted in a hypertonic ionic strength solution prior to polymerization (e.g., a high glucose solution, e.g., Dulbecco's Modified Eagle Medium (DMEM)). In some embodiments, the biocompatible scaffold is bioresorbable. In some embodiments, the biocompatible scaffold includes a synthetic heparin mimetic. In particular, the synthetic polymer of the invention may include an amount of negative charge that, in some embodiments, is similar to the amount of negative charge present in heparin. Accordingly, the synthetic polymer of the disclosure can mimic the functional properties of heparin. For example, the synthetic polymer of the disclosure has the potential to bind various bioactive agents, e.g., growth factors, that naturally bind to heparin. Therefore, the synthetic polymer of the disclosure, as well as the hydrogel comprising the synthetic polymer described herein can bind various bioactive agents, e.g., growth factors, thereby preventing the bioactive agents from diffusing away and maintaining the bioactive agents at a high concentration locally, so that they can act on cells and promote various cell functions.

[0187] In some embodiments, the engineered tissue construct includes between 1 x 106cells / mL and 1 x 108cells / mL, e.g., from 1 x 106cells / mL to 10 x 106cells / mL (e.g., 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 5 x 106cells / mL, 6 x 106cells / mL, 7 x 106cells / mL, 8 x 106cells / mL, 9 x

[0188] 106cells / mL, or 1 x 107cells / mL) or from 1 x 107cells / mL to 1 x 108cells / mL (e.g., 2 x 107cells / mL, 3 x

[0189] 107cells / mL, 4 x 107cells / mL, 5 x 107cells / mL, 6 x 107cells / mL, 7 x 107cells / mL, 8 x 107cells / mL, 9 x 107cells / mL, or 1 x 108cells / mL).

[0190] In some embodiments, the engineered tissue construct is from 0.1 mL to 5 L (e.g., 0.2 mL to 5 L, 0.3 mL to 5 L, 0.4 mL to 5 L, 0.5 mL to 5 L, 1 mL to 5 L, 5 mL to 5 L, 10 mL to 5 L, 100 mL to 5 L, 1 L to 5 L, 2 L to 5 L, 3 L to 5 L, or 4 L to 5 L) in volume. In some embodiments, the engineered tissue construct has a volume of between 20 mL and 1 .2 L, e.g., from 20 mL to 100 mL (e.g., 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL), from 100 mL to 500 mL (e.g., 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL), or from 500 mL to 1 .2 L (e.g., 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 1 .1 L, or 1 .2 L). For example, in some embodiments, the engineered tissue construct is from 0.2 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.3 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.4 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.5 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 1 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 5 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 10 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 100 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 1 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 2 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 3 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 4 mL to 5 L in volume. In some embodiments, the engineered tissue construct further includes a reinforcing agent. In some embodiments the reinforcing agent is selected from the list including fibrin, surgical mesh, alginate, collagen, polyethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic) acid (PLGA), and poly (l-lactic acid) (PLLA). In some embodiments the reinforcing agent is fibrin. In some embodiments the reinforcing agent is surgical mesh. In some embodiments the reinforcing agent is alginate. In some embodiments the reinforcing agent is collagen. In some embodiments the reinforcing agent is polyethylene glycol). In some embodiments the reinforcing agent is PVDA. In some embodiments the reinforcing agent is PVDF. In some embodiments the reinforcing agent is PLGA. In some embodiments the reinforcing agent is PLLA. In some embodiments the reinforcing agent is any suitable agent.

[0191] In some embodiments, the engineered tissue construct has a serpentine topography (e.g., to increase surface area).

[0192] Fibroblasts

[0193] Any suitable fibroblast cell is contemplated within the scope of the disclosure described herein, e.g., within a cell aggregate or engineered tissue construct. In some embodiments, the fibroblasts are human fibroblasts (e.g., dermal fibroblasts, e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts). In some embodiments, the fibroblasts are human dermal fibroblasts. In some embodiments, the fibroblasts are normal human dermal fibroblasts. In some embodiments, the fibroblasts are neonatal foreskin fibroblasts. In some embodiments, the fibroblasts human lung fibroblasts. In some embodiments, the fibroblasts are human ventricular cardiac fibroblasts. In some embodiments, the fibroblasts are human atrial cardiac fibroblasts. In some embodiments, the fibroblasts are human uterine fibroblasts. In some embodiments, the fibroblasts are human bladder fibroblasts. In some embodiments, the fibroblasts are human gingival fibroblasts. In some embodiments, the fibroblasts are human pericardial fibroblasts. In some embodiments, the fibroblasts are human gall bladder fibroblasts. In some embodiments, the fibroblasts are human portal vein fibroblasts. In some embodiments, the fibroblasts are vas deferens fibroblasts.

[0194] In some embodiments, a population of fibroblasts is engineered with an exogenous polynucleotide. In some embodiments, the population of fibroblasts includes from 6 x 103to 1 .8 x 1012(e.g., from 1 to 1 .8 x 1012, from 10 to 1 .8 x 1012, from 100 to 1 .8 x 1012, from 1 x 103to 1 .8 x 1012, from 2 x 103to 1 .8 x 1012, from 3 x 103to 1 .8 x 1012, from 4 x 103to 1 .8 x 1012, from 5 x 103to 1 .8 x 1012, from 6 x

[0195] 103to 1 .8 x 1012, from 7 x 103to 1 .8 x 1012, from 8 x 103to 1 .8 x 1012, from 9 x 103to 1 .8 x 1012, from 1 x

[0196] 104to 1 .8 x 1012, from 2 x 104to 1 .8 x 1012, from 3 x 104to 1 .8 x 1012, from 4 x 104to 1 .8 x 1012, from 5 x

[0197] 104to 1 .8 x 1012, from 6 x 104to 1 .8 x 1012, from 7 x 104to 1 .8 x 1012, from 8 x 104to 1 .8 x 1012, from 9 x

[0198] 104to 1 .8 x 1012, from 1 x 105to 1 .8 x 1012, from 2 x 105to 1 .8 x 1012, from 3 x 105to 1 .8 x 1012, from 4 x

[0199] 105to 1 .8 x 1012, from 5 x 105to 1 .8 x 1012, from 6 x 105to 1 .8 x 1012, from 7 x 105to 1 .8 x 1012, from 8 x

[0200] 105to 1 .8 x 1012, from 9 x 105to 1 .8 x 1012, from 1 x 106to 1 .8 x 1012, from 2 x 106to 1 .8 x 1012, 3 x 106to 1 .8 x 1012, 4 x 106to 1 .8 x 1012, 5 x 106to 1 .8 x 1012, 6 x 106to 1 .8 x 1012, 7 x 106to 1 .8 x 1012, 8 x 106to 1 .8 x 1012, 9 x 106to 1 .8 x 1012, from 1 x 107to 1 .8 x 1012, from 2 x 107to 1 .8 x 1012, from 3 x 107to 1 .8 x

[0201] 1012, from 4 x 107to 1 .8 x 1012, from 5 x 107to 1 .8 x 1012, from 6 x 107to 1 .8 x 1012, from 7 x 107to 1 .8 x

[0202] 1012, from 8 x 107to 1 .8 x 1012, from 9 x 107to 1 .8 x 1012, from 1 x 108to 1 .8 x 1012, from 2 x 108to 1 .8 x

[0203] 1012, from 3 x 108to 1 .8 x 1012, from 4 x 108to 1 .8 x 1012, from 5 x 108to 1 .8 x 1012, from 6 x 108to 1 .8 x

[0204] 1012, from 7 x 108to 1 .8 x 1012, from 8 x 108to 1 .8 x 1012, from 9 x 108to 1 .8 x 1012, from 1 x 109to 1 .8 x

[0205] 1012, from 2 x 109to 1 .8 x 1012, from 3 x 109to 1 .8 x 1012, from 4 x 109to 1 .8 x 1012, from 5 x 109to 1 .8 x

[0206] 1012, from 6 x 109to 1 .8 x 1012, from 7 x 109to 1 .8 x 1012, from 8 x 109to 1 .8 x 1012, from 9 x 109to 1 .8 x

[0207] 1012, from 1 x 1010to 1 .8 x 1012, from 2 x 1010to 1 .8 x 1012, from 3 x 1010to 1 .8 x 1012, from 4 x 1010to 1 .8 x 1012, from 5 x 1010to 1 .8 x 1012, from 6 x 1010to 1 .8 x 1012, from 7 x 1010to 1 .8 x 1012, from 8 x 1010to 1 .8 x 1012, from 9 x 1010to 1 .8 x 1012, from 1 x 1011to 1 .8 x 1012, from 2 x 1011to 1 .8 x 1012, from 3 x 1011to 1 .8 x 1012, from 4 x 1011to 1 .8 x 1012, from 5 x 1011to 1 .8 x 1012, from 6 x 1011to 1 .8 x 1012, from 7 x 1011to 1 .8 x 1012, from 8 x 1011to 1 .8 x 1012, from 9 x 1011to 1 .8 x 1012, or from 1 x 1012to 1 .8 x 1012) fibroblasts.

[0208] Aggregated Hepatocytes and Fibroblasts

[0209] The cellular compositions disclosed herein can be provided as a suspension containing hepatocytes and fibroblasts. In some embodiments, the population of hepatocytes and the fibroblasts are aggregated, e.g., in spheroids.

[0210] In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :10 and 4:1 (e.g., 1 :10 and 4:1 , 1 :10 and 3:1 , 1 :10 and 2:1 , 1 :10 and 1 :1 , 1 :9 and 4:1 , 1 :9 and 3:1 , 1 :9 and 2:1 , 1 :9 and 1 :1 , 1 :8 and 4:1 , 1 :8 and 3:1 , 1 :8 and 2:1 , 1 :8 and 1 :1 , 1 :7 and 4:1 , 1 :7 and 3:1 , 1 :7 and 2:1 , 1 :7 and 1 :1 , 1 :6 and

[0211] 4:1 , 1 :6 and 3:1 , 1 :6 and 2:1 , 1 :6 and 1 :1 , 1 :5 and 4:1 , 1 :5 and 3:1 , 1 :5 and 2:1 , 1 :5 and 1 :1 , 1 :4 and 4:1 ,

[0212] 1 :4 and 3:1 , 1 :4 and 2:1 , 1 :4 and 1 :1 , 1 :3 and 4:1 , 1 :3 and 3:1 , 1 :3 and 2:1 , 1 :3 and 1 :1 , 1 :2 and 4:1 , 1 :2 and 3:1 , 1 :2 and 2:1 , 1 :2 and 1 :1 , 1 :1 and 4:1 , 1 :1 and 3:1 , 1 :1 and 2:1 , and 1 :0 and 1 :1 ).

[0213] For example, in some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :9 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :8 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :7 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :6 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :5 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :4 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :3 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :2 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :1 and 4:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :0 and 4:1 .

[0214] In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :10 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :9 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :8 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :7 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :6 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :5 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :4 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :3 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :2 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :1 and 3:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :0 and 3:1 .

[0215] In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :10 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :9 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :8 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :7 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :6 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :5 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :4 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :3 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :2 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :1 and 2:1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :0 and 2:1 .

[0216] In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :10 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :9 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :8 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :7 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :6 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :5 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :4 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :3 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :2 and 1 :1 . In some embodiments, the ratio of hepatocytes to fibroblasts is between 1 :1 and 1 :0.

[0217] In some embodiments, the population of hepatocytes and the fibroblasts are aggregated in a biocompatible scaffold. For example, in some embodiments, the population of hepatocytes and the fibroblasts are aggregated in spheroids, and the spheroids are distributed non-homogenously, in a layer, along the z-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the x-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the y-axis of the biocompatible scaffold.

[0218] In some embodiments, the hepatocytes and fibroblasts are in a biocompatible scaffold, and the population of hepatocytes and the population of fibroblasts together account for at least 70% (e.g., at least 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, or 100%) of the total cells in the engineered tissue construct. For example, in some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 71% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 72% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 73% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 74% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 75% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 80% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 85% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 90% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for at least 95% of the total cells in the engineered tissue construct. In some embodiments, the population of hepatocytes and the population of fibroblasts together account for 100% of the total cells in the engineered tissue construct.

[0219] In some embodiments, the hepatocytes and fibroblasts are distributed non-homogenously along the z-axis of the biocompatible scaffold.

[0220] In some embodiments, the hepatocytes and fibroblasts are distributed homogenously along the x- axis of the biocompatible scaffold.

[0221] In some embodiments, the hepatocytes and fibroblasts are distributed homogenously along the y- axis of the biocompatible scaffold.

[0222] In some embodiments, the layer of hepatocyte and fibroblasts aggregates in the biocompatible scaffold is from 100 pm to 1 mm (e.g., 200 pm to 900 pm, 300 pm to 800 pm, 400 pm to 700 pm, or 500 pm to 600 pm) thick. For example, in some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 200 pm to 900 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 300 pm to 800 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 400 pm to 700 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 500 pm to 600 pm thick.

[0223] In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 100 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 200 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 300 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 400 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 500 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 600 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 700 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 800 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 900 pm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 1 mm thick. In some embodiments, the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 2 mm thick.

[0224] In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.06 M / cm2to 150 M / cm2(e.g., 0.07 M / cm2to 149 M / cm2, 0.08 M / cm2to 148 M / cm2, 0.09 M / cm2to 147 M / cm2, 0.1 M / cm2to 146 M / cm2, 0.2 M / cm2to 145 M / cm2, 0.3 M / cm2to 140 M / cm2, 0.4 M / cm2to 130 M / cm2, 0.5 M / cm2to 120 M / cm2, 1 M / cm2to 1 10 M / cm2, 2 M / cm2to 100 M / cm2, 3 M / cm2to 50 M / cm2, 4 M / cm2to 40 M / cm2, 5 M / cm2to 30 M / cm2, or 10 M / cm2to 20 M / cm2). For example, in some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.07 M / cm2to 149 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.08 M / cm2to 148 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.09 M / cm2to 147 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.1 M / cm2to 146 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.2 M / cm2to 145 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.3 M / cm2to 140 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.4 M / cm2to 130 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 0.5 M / cm2to 120 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 1 M / cm2to 1 10 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 2 M / cm2to 100 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 3 M / cm2to 50 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 4 M / cm2to 40 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 5 M / cm2to 30 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is from 10 M / cm2to 20 M / cm2.

[0225] In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.06 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.07 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.08 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.09 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.1 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.2 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.3 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.4 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 0.5 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 1 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 2 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 3 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 4 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 5 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 10 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 20 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 30 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 40 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 50 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 100 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 110 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 120 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 130 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 140 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 145 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 146 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 147 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 148 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 149 M / cm2. In some embodiments, the density of hepatocytes in the layer of hepatocyte and fibroblast aggregates in the biocompatible scaffold is 150 M / cm2.

[0226] In some embodiments, the thickness of the layer is dependent upon the hepatocyte density.

[0227] Other Cell Types

[0228] The engineered tissue construct may include other cell types. In some embodiments, the engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of other cell types. For example, other cell types may include stellate cells, Kupffer cells, pit cells, cholangiocytes, portal fibroblasts, and liver sinusoidal endothelial cells. In some embodiments, the engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of stellate cells. In some embodiments, the engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of Kupffer cells. In some embodiments, the engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of pit cells. In some embodiments, the engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of bile duct cells.

[0229] In some embodiments, the engineered tissue construct includes endothelial cells. In some embodiments, the engineered tissue construct includes up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 1 %) of endothelial cells. For example, in some embodiments, the engineered tissue construct includes up to 29% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 28% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 27% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 26% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 25% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 20% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 15% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 10% of endothelial cells. In some embodiments, the engineered tissue construct includes up to 1% of endothelial cells. In some embodiments, the population of endothelial cells is arranged as one or more cords.

[0230] Biocompatible Hydrogel Scaffolds

[0231] The compositions disclosed herein can be provided as a suspension in a biocompatible scaffold containing the hepatocytes or hepatocyte progenitor cells. In some embodiments, the population of hepatocytes or hepatocyte progenitor cells are aggregated in spheroids. In some embodiments, the biocompatible scaffold has an x-axis, a y-axis, and a z-axis. For example, in some embodiments, the population of hepatocytes or hepatocyte progenitor cells are aggregated in spheroids and the spheroids are distributed non-homogenously, in a layer, along the z-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the x-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the y-axis of the biocompatible scaffold.

[0232] The biocompatible scaffold may be liquid, gel, semi-solid, or solid at room temperature (e.g., 25 °C). The biocompatible scaffold may be biodegradable or non-biodegradable. In some embodiments, the scaffold is bioresorbable or bioreplaceable. In some embodiments, the scaffold includes fibrin (e.g., human fibrin or human fibrinogen, e.g., FIBRYGA®). Exemplary biocompatible scaffolds include polymers and hydrogels include collagen, fibrinogen, fibrin, chitosan, MATRIGEL™, dextrans including chemically cross-linkable or photo-cross-linkable dextrans, processed tissue matrix such as submucosal tissue, PEG hydrogels (e.g., heparin-conjugated PEG hydrogels), poly(lactic-co-glycolic acid) (PLGA), hydroxyethyl methacrylate (HEMA), gelatin, alginate, agarose, polysaccharides, hyaluronic acid (HA), peptide-based self-assembling gels, thermo-responsive poly(NIPAAm). A number of biopolymers are known to those skilled in the art (Bryant and Anseth, J. Biomed. Mater. Res. 59(1 ):63-72, 2002; Mann et al., Biomaterials 22(22): 3045-3051 , 2001 ; Mann et al., Biomaterials. 22 (5):439-444, 2001 , and Peppas et al., Eur. J. Pharm. Biopharm. 50(1 ), 27-46, 2000; all incorporated by reference). In other embodiments, the biocompatible scaffold may contain a biopolymer having any of a number of growth factors, adhesion molecules, degradation sites or bioactive agents to enhance cell viability or for any of a number of other reasons. Such molecules are well known to those skilled in the art.

[0233] In some embodiments, the PEG hydrogel may be chemically cross-linkable and / or modified with bifunctional groups.

[0234] In certain embodiments, the biocompatible scaffold includes allogeneic components, autologous components, or both allogeneic components and autologous components. In certain embodiments, the biocompatible scaffold includes synthetic or semi-synthetic materials. In certain embodiments, the biocompatible scaffold includes a framework or support, such as a fibrin-derived scaffold.

[0235] In some embodiments, the biocompatible scaffold is fibrin. Biocompatible hydrogel scaffolds suitable for use include any polymer that is gellable in situ, e.g., one that does not require chemicals or conditions (e.g., temperature or pH) that are not cytocompatible. This includes both stable and biodegradable biopolymers.

[0236] Polymers for use herein are preferably crosslinked, for example, ionically crosslinked. In certain embodiments, the methods and constructs described herein use polymers in which polymerization can be promoted photochemically (i.e., photo crosslinked), by exposure to an appropriate wavelength of light (i.e., photopolymerizable) or a polymer which is weakened or rendered soluble by light exposure or other stimulus. Although some of the polymers listed above are not inherently light sensitive (e.g., collagen, HA), they may be made light sensitive by the addition of acrylate or other photosensitive groups.

[0237] In certain embodiments, the method utilizes a photoinitiator. A photoinitiator is a molecule that is capable of promoting polymerization of hydrogels upon exposure to an appropriate wavelength of light as defined by the reactive groups on the molecule. In the context of the disclosure, photoinitiators are cytocompatible. A number of photoinitiators are known that can be used with different wavelengths of light. For example, 2,2-dimethoxy-2-phenyl-acetophenone, HPK 1 -hydroxycyclohexyl-phenyl ketone and Irgacure 2959 (hydroxyl-1 -[4-(hydroxyethoxy)phenyl]-2methyl-1 propanone) are all activated with UV light (365 nm). Other crosslinking agents activated by wavelengths of light that are cytocompatible (e.g., blue light) can also be used with the methods described herein.

[0238] In other embodiments, the method involves the use of polymers bearing non-photochemically polymerizable moieties. In certain embodiments, the non-photochemically polymerizable moieties are Michael acceptors. Non-limiting examples of such Michael acceptor moieties include a,p-unsaturated ketones, esters, amides, sulfones, sulfoxides, phosphonates. Additional non-limiting examples of Michael acceptors include quinines and vinyl pyridines. In some embodiments, the polymerization of Michael acceptors is promoted by a nucleophile. Suitable nucleophiles include, but are not limited to thiols, amines, alcohols, and molecules possessing thiol, amine, and alcohol moieties. In certain embodiments, the disclosure features use of thermally crosslinked polymers.

[0239] In some embodiments, the z-axis of the biocompatible scaffold is from 500 pm to 5 mm (e.g., 600 pm to 4 mm, 700 pm to 3 mm, 800 pm to 2 mm, or 900 pm to 1 mm). For example, in some embodiments, the z-axis of the biocompatible scaffold is from 600 pm to 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 700 pm to 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 800 pm to 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 900 pm to 1 mm.

[0240] In some embodiments, the z-axis of the biocompatible scaffold is 500 pm. In some embodiments, the z-axis of the biocompatible scaffold is 600 pm. In some embodiments, the z-axis of the biocompatible scaffold is 700 pm. In some embodiments, the z-axis of the biocompatible scaffold is 800 pm. In some embodiments, the z-axis of the biocompatible scaffold is 900 pm. In some embodiments, the z-axis of the biocompatible scaffold is 1 mm. In some embodiments, the z-axis of the biocompatible scaffold is 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is 5 mm.

[0241] In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer of cell aggregates, e.g., hepatocytes, in the biocompatible scaffold is from 20:1 to 1 :1 (e.g., 19:1 to 1 :1 , 18:1 to 1 :1 , 17:1 to 1 :1 , 16:1 to 1 :1 , 15:1 to 1 :1 , 14:1 to 1 :1 , 13:1 to 1 :1 , 12:1 to 1 :1 , 11 :1 to 1 :1 , 10:1 to 1 :1 , 9:1 to 1 :1 , 8:1 to 1 :1 , 7:1 to 1 :1 , 6:1 to 1 :1 , 5:1 to 1 :1 , 4:1 to 1 :1 , 3:1 to 1 :1 , or 2:1 to 1 :1 ). For example, in some embodiments the ratio of height of the biocompatible scaffold to height of the layer is from 19:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 18:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 17:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 16:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 15:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 14:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 13:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 12:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 11 :1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 10:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 9:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 8:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 7:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 6:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 5:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 4:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 3:1 to 1 :1 .

[0242] In some embodiments, the biocompatible scaffold includes a synthetic heparin mimetic. In particular, the synthetic polymer of the invention may include an amount of negative charge that, in some embodiments, is similar to the amount of negative charge present in heparin. Accordingly, the synthetic polymer of the disclosure can mimic the functional properties of heparin. For example, the synthetic polymer of the disclosure has the potential to bind various bioactive agents, e.g., growth factors, that naturally bind to heparin. Therefore, the synthetic polymer of the disclosure, as well as the hydrogel comprising the synthetic polymer described herein can bind various bioactive agents, e.g., growth factors, thereby preventing the bioactive agents from diffusing away and maintaining the bioactive agents at a high concentration locally, so that they can act on cells and promote various cell functions.

[0243] Methods for Making Engineered Tissue Constructs

[0244] The disclosure also features methods for making an engineered tissue construct containing hepatocytes or hepatocyte progenitor cells (e.g., hepatocytes, e.g., primary human hepatocytes (PHH)). The engineered tissue construct may optionally further include fibroblasts (e.g., dermal fibroblast, e.g., human dermal fibroblasts e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts). Frozen master cell banks (MCB) may be sourced through external suppliers and may be received as cryopreserved cells. In some examples, all cell types are terminally differentiated cells isolated from primary donors obtained with appropriate donor consent for therapeutic use. For example, hepatocytes (e.g., PHH) may be obtained from cadaveric donors via collagenase perfusion, Percoll density gradient purification, and subsequent cryopreservation to create an MCB. Hepatocytes may be stored cryopreserved until initiation of a manufacturing build. Prior to accepting the lot as a released MCB, release testing is conducted on hepatocyte (e.g., PHH) candidate MCBs to establish that their performance characteristics meet acceptance criteria for characterization, release, and stability. Fibroblasts (e.g., dermal fibroblasts, e.g., human dermal fibroblasts e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts) may be, for example, isolated from a single donor of neonatal foreskin by physical separation of dermal and epidermal layers and sequential digestion with dispase and collagenase. After isolation, fibroblasts may be minimally expanded and cryopreserved to create a frozen MCB. Frozen MCBs may be shipped to the manufacturing site and fibroblasts are expanded to create working cell banks (WCB), which may be then cryopreserved until initiation of a manufacturing build. These WCB may be released based on specific acceptance criteria prior to use in the manufacturing process.

[0245] Upon initiation of a manufacturing build, fibroblasts (e.g., human dermal fibroblasts e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts) may be thawed from their respective WCB expanded, and tested to measure viability and cell count. Hepatocytes (e.g., PHH) may be thawed from the hepatocyte MCB and tested to measure viability and cell count prior to optionally being combined at a ratio (e.g., 1 :2) with fibroblasts, centrifuged into arrays of microwells (e.g., pyramidal microwells), and incubated for 2-3 days to promote self-assembly of the cells into multicellular hepatic aggregates (e.g., spheroidal aggregates). Aggregates (e.g., spheroidal aggregates) may be deemed acceptable for encapsulation after microscopic confirmation of compaction. Alternatively, a population of hepatocytes may be encapsulated without fibroblasts.

[0246] The cell aggregates may then be encapsulated with a solution (e.g., a fibrinogen solution) that is polymerized (e.g., with thrombin). These encapsulation steps may occur within a mold (e.g., a cylindrical mold) that controls the overall dimensions of the engineered tissue construct to be 500 pm to 5 mm in thickness and with an outer diameter of 6 mm to 100 cm (e.g., 7 mm to 999 mm, 8 mm to 998 mm, 9 mm to 997 mm, 10 mm to 996 mm, 20 mm to 995 mm, 30 mm to 990 mm, 40 mm to 980 mm, 60 mm to 960 mm, 90 mm to 930 mm, 100 mm to 900 mm, 200 mm to 800 mm, 300 mm to 700 mm, 400 mm to 600 mm, or 500 mm). The thickness may be controlled by the volume of cell-hydrogel suspension and targeted to be 2 mm in thickness.

[0247] In some embodiments, the mold may be any shape (e.g., cylindrical, square, or square with rounded corners).

[0248] Within the solution (e.g., a fibrinogen solution that is polymerized e.g., with thrombin), the hepatocyte cell aggregates are allowed to non-homogenously distribute (e.g., by gravity) along the z-axis of the biocompatible scaffold into a layer (e.g., to settle), thereby forming a one-sided engineered tissue construct.

[0249] In some embodiments, two or more of the one-sided engineered tissue constructs are assembled with each of the layers facing outwardly, respectively, thereby forming a two-sided engineered tissue construct.

[0250] The engineered tissue constructs of the present disclosure can be formed by a process described herein. In some embodiments, engineered tissue constructs with defined cellular configurations in a biocompatible hydrogel scaffold may be prepared by photopatterning PEG hydrogels containing the hepatocyte cell populations, resulting in a hydrogel network consisting of 3D cell hepatocytes, and optionally, hepatocytes. Further control of cell orientation within these patterned domains may be achieved utilizing dielectrophoretic patterning techniques. Dielectrophoresis (DEP) can be used alone for patterning of cells in relatively homogeneous slabs of hydrogel or in conjunction with the photopolymerization method.

[0251] In some embodiments, organizing cells and material into spatial arrangements, such as engineered tissue constructs, can be accomplished by physically constraining the placement of cells / material by the use of wells or grooves, or injecting cells into microfluidic channels or oriented void spaces / pores. In certain embodiments, the cells can be organized by physically positioning cells with electric fields, magnetic tweezers, optical tweezers, ultrasound waves, pressure waves, or micromanipulators. In some embodiments, the population of cells (e.g., hepatocytes or hepatocytes and fibroblasts) are aggregated in spheroids and the spheroids are allowed (e.g., by gravity) to non- homogenously distribute along the z-axis of the biocompatible scaffold into a layer

[0252] In certain embodiments, the method for fabricating engineered tissue constructs and embedding the constructs in extracellular matrix includes (1 ) generating 3D templates that have been defined with channels or trenches, (2) suspending the population of cells and the population of cells in liquid collagen and centrifuging these cells into the channels of the template, (3) removing excess cell / collagen suspension to allow aggregates to form, and (4) removing aggregates from templates via encapsulation in an extracellular matrix scaffold.

[0253] In some embodiments, the method for fabricating the engineered tissue constructs includes (1 ) suspending the population of cells in a naturally derived and / or synthetic scaffolding, (2) placing the suspended cells into the channels of a 3D template, and (3) allowing the cells to form one or more aggregates at least partially embedded in the naturally derived and / or synthetic scaffolding. In some embodiments, the 3D template can be generated by molding, templating, photolithography, printing, deposition, sacrificial molding, stereolithography, or a combination thereof.

[0254] In some embodiments, an engineered tissue construct can be fabricated through the use a custom 3D printer technology to extrude lattices of carbohydrate glass filaments with predefined diameters, spacings and orientations. For example, in some embodiments, soluble (clinical-grade, sterile) fibrinogen and thrombin are combined and poured over the lattice. After the solution has polymerized into insoluble fibrin, the carbohydrate filaments are dissolved, leaving behind channels within the fibrin. The channels can then be filled with a suspension of cells in a naturally derived or synthetic scaffolding (e.g., soluble type I collagen) that subsequently is polymerized to trap the cells within the channels. The methods allow for the formation of three-dimensional scaffolds from hundreds of micrometers to tens of centimeters in length and width, and tens of micrometers to hundreds of micrometers in height. A resolution of up to 100 micrometers in the photopolymerization method and possible single cell resolution (10 pm) in the DEP method is achievable. Photopolymerization apparatus, DEP apparatus, and other methods to produce 3-dimensional co-cultures are described in U.S. Pat. No. 8,906,684, which is incorporated herein by reference.

[0255] The cells can be cultured in vitro under various culture conditions. The cells (e.g., primary cells) can be expanded in culture, e.g., grown under conditions that promote their proliferation. Culture medium can be liquid or semi-solid, e.g., containing agar, methylcellulose, and the like. The cell population can be suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI 1640, normally supplemented with fetal calf serum (5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g., penicillin and streptomycin. The culture may contain growth factors to which the regulatory T cells are responsive. Growth factors, as defined herein, can be molecules capable of promoting survival, growth and / or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.

[0256] The cells produced by the methods described herein can be used immediately in the making of an engineered tissue construct. Alternatively, the cells can be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. For example, the cells can be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0257] Implantation of Hepatocytes or Engineered Tissue Constructs

[0258] Described herein is a method for implanting hepatocytes or hepatocyte progenitor cells or an engineered tissue construct that includes a population of hepatocytes or hepatocyte progenitor cells (e.g., engineered to secrete an incretin, e.g., GLP-1 RA) in a biocompatible scaffold. An engineered tissue construct may further include a population of fibroblasts. In some embodiments, upon implantation, the population of cells are engrafted and vascularized in the subject. In some embodiments, the method treats a disease or disorder in a subject. In some embodiments, the disease or disorder is diabetes (e.g., type 2 diabetes) or obesity. For example, the hepatocytes or hepatocyte progenitor cells may secrete the incretin to impart a therapeutic effect, e.g., by increasing satiety, enhancing insulin secretion, and / or reducing blood sugar.

[0259] In some embodiments, the method treats a cardiovascular disease (e.g., high blood pressure (hypertension), arrhythmia, valve disease, coronary artery disease, heart failure, peripheral artery disease, aortic disease, pericardial disease, cerebrovascular disease, deep vein thrombosis (DVT), or a congenital heart disease), a neurodegenerative disease (e.g., dementia, Alzheimer’s disease, frontotemporal dementia, chronic traumatic encephalopathy, Lewy body dementia, multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), or prion disease), a metabolic dysfunction- associated steatohepatitis (e.g., non-alcoholic steatohepatitis (NASH), or another liver condition). In some embodiments, the engineered tissue construct is implanted in a human subject in an extraperitoneal space, in an extrapleural space, or on a liver surface. In some embodiments, the engineered tissue construct is implanted in an extraperitoneal space, in an extrapleural space, on a surface of the liver, in a muscle site (e.g., on a surface of a muscle, within a muscle sheath, or beneath a muscle), in a pleural space, in an omentum site, in a subcutaneous site, on a surface of the pancreas, on a surface of the spleen, on a surface of the kidney, in a bone marrow site, in a bursa site, in a peritoneal site (e.g., mesentery), or in a lesser sac site.

[0260] The engineered tissue constructs described herein can be implanted in a subject. Non-limiting examples of nonhuman subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, fowl, pigs, horses, cows, goats, or sheep. In certain embodiments, the subject can be any animal. In certain embodiments, the subject can be any mammal. In certain embodiments, the subject can be a human.

[0261] In some embodiments, the subject has an age of between 1 day and 120 years (e.g., between 1 day and 7 days, between 1 day and 1 month, between 1 day and 6 months, between 1 day and 1 year, between 1 day and 2 years, between 1 month and 12 months, between 1 month and 6 months, between 6 months and 2 years, between 1 year and 2 years, between 1 year and 5 years, between 1 year and 10 years, between 10 years and 20 years, between 10 years and 50 years, between 50 years and 80 years, or between 60 years and 90 years). In some embodiments, the subject has an age of between 1 day and 1 year. In some embodiments, the subject is a newborn or an infant.

[0262] In some embodiments, the engineered tissue construct is implanted into a subject in an extraperitoneal space, in an extrapleural space, on a surface of the liver, in a muscle site (e.g., a surface of a muscle, within a muscle sheath, or beneath a muscle), in a pleural space, in an omentum site, in a subcutaneous site, on a surface of the pancreas, on a surface of the spleen, on a surface of the kidney, in a bone marrow site, in a bursa site, in a peritoneal site, or in a lesser sac site

[0263] In some embodiments, the engineered tissue construct is implanted into the subject in an extraperitoneal space, in an extrapleural space, or on a liver surface. In some embodiments, the engineered tissue construct is implanted in the extraperitoneal space. In some embodiments, the engineered tissue construct is implanted in a pre-peritoneal space, a retroperitoneal space, or a subperitoneal space.

[0264] In some embodiments, the engineered tissue construct is implanted on the surface of the liver.

[0265] In some embodiments, the engineered tissue construct is layered on the dome of the liver and / or covered with omentum.

[0266] In some embodiments, the muscle site is a surface of a muscle. In some embodiments, the muscle site is within a muscle sheath. In some embodiments, the muscle site is beneath a muscle.

[0267] In some embodiments, the omentum site includes an omentum pedicle flap, an omentum free flap, an omental bursa, or the omentum in situ.

[0268] In some embodiments, the engineered tissue construct is implanted subcutaneously with an omental flap, subcutaneously with an adjuvant, or subcutaneously with an arteriovenous fistula.

[0269] In some embodiments, the engineered tissue construct is implanted into the subject in an implantation site selected from the group consisting of the peritoneum, an extraperitoneal site (e.g., retroperitoneum), pre-perintoneal space, or a subperitoneal space), peritoneal cavity (e.g., omentum or mesentery), rectus abdominis muscle, abdominal oblique muscle, quadriceps femoris muscle, gluteus maximus, a hamstring muscle (e.g., a semimembranosus, a semitendinosus, or a biceps femoris), deltoid, biceps, latissimus dorsi, extraperitoneal fat, and renal capsule; an extraperitoneal site, a site on the surface of the liver, or an extrapleural site; or a site that is suitable for neovascularization. For example, in some embodiments, the peritoneum is the retroperitoneum. In some embodiments, the peritoneal cavity is the omentum. In some embodiments, the peritoneal cavity is the mesentery. In some embodiments, the omentum is the greater omentum or the omental bursa. In some embodiments, the mesentery is the small intestinal mesentery. In some embodiments, the engineered tissue construct is implanted into the subject as a pedicled omental wrap or an omental wrap.

[0270] In some embodiments, one or more engineered tissue constructs is implanted in one or more implantation sites selected from the group consisting of the peritoneum, retroperitoneum, peritoneal cavity (e.g., omentum or mesentery), rectus abdominis muscle, abdominal oblique muscle (including an internal oblique muscle or an external oblique muscle), transversus abdominis muscle, quadriceps femoris muscle, gluteus maximus, a hamstring muscle (e.g., a semimembranosus muscle, a semitendinosus muscle, or a biceps femoris muscle), deltoid , biceps, latissimus dorsi, extraperitoneal fat, and renal capsule; an extraperitoneal site, a surface of the liver, or an extrapleural site; or a site that is suitable for neovascularization. For example, in some embodiments, the engineered tissue construct is implanted in the retroperitoneum. For example, in some embodiments, two engineered tissue constructs are implanted bilaterally on an omentum site.

[0271] The engineered tissue construct can be implanted in any suitable manner, often with pharmaceutically acceptable carriers. In some embodiments, the engineered tissue construct is implanted on a surface of a tissue or organ. In some embodiments, the engineered tissue construct is implanted on an orthotopic site. In other embodiments, the engineered tissue construct is implanted on an ectopic site.

[0272] Any suitable approach may be used to perform the implantation. For example, the engineered tissue construct may be implanted using an open surgical procedure or minimally invasive surgery. In some embodiments, the engineered tissue construct may be implanted using an open surgical procedure. In other embodiments, the engineered tissue construct may be implanted using a minimally invasive surgery.

[0273] In some embodiments, the methods of implantation include dissecting the site of implantation. In some embodiments, the methods of implantation include forming a pocket for the implant. For example, the site of implantation may be dissected down through skin, fat, connective tissue and / or muscle, e.g., to the peritoneum. Dissection (e.g., blunt dissection) of connective tissue, e.g., between the peritoneum and muscle, may be performed to form an implant pocket. One or more sutures may be added to mechanically stabilize the graft at the desired site of implantation, e.g., in the pocket.

[0274] The engineered tissue construct may be affixed to the subject using any suitable approach. For example, the engineered tissue construct may be affixed using sutures, staples, or by welding (e.g., laser tissue welding). In some embodiments, the engineered tissue construct is affixed by one or more sutures or one or more staples. In some embodiments, the engineered tissue construct is affixed by suturing adjoining tissue to restrain migration of the engineered tissue construct. For example, in some embodiments, the engineered tissue construct is implanted at an extraperitoneal site, and the engineered tissue construct is affixed by suturing the muscle fascia to the peritoneum at one or more positions surrounding the engineered tissue construct. In some embodiments, the engineered tissue construct is not directly sutured or stapled (in other words, the sutures or staples do not penetrate the engineered tissue construct itself). In some embodiments, the engineered tissue construct is implanted in a site that is suitable for neovascularization. In some embodiments, a site that it suitable for neovascularization is one having a microvessel density of from 3.6 vessels / mm2to 4500 vessels / mm2(e.g., 3.7 vessels / mm2to 4000 vessels / mm2, 3.8 vessels / mm2to 3500 vessels / mm2, 3.9 vessels / mm2to 3000 vessels / mm2, 4 vessels / mm2to 2500 vessels / mm2, 5 vessels / mm2to 2000 vessels / mm2, 10 vessels / mm2to 1000 vessels / mm2, or 100 vessels / mm2).

[0275] In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 3.7 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4.5 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 5 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 10 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 50 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 100 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 1000 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4500 vessels / mm2. Autologous, allogenic or xenogenic cells may be used. The cells may be implanted in any physiologically acceptable medium. In one embodiment, the cells are cryopreserved in 5-20% DMSO, 5% dextrose and autologous serum. As is familiar to those of skill in the art, dosage of the cells of the present invention to be implanted in vivo is determined with reference to various parameters, including the species of the host, the age, weight, and disease status. Dosage also depends upon the location to be targeted within the subject. For example, implantation of the engineered tissue construct into the omentum may require different dosages than implantation to the mesentery. The dosage is preferably chosen so that implantation causes an effective result, which can be measured by molecular assays (e.g., a liver function test) or by monitoring a suitable symptom in the subject.

[0276] In some embodiments, the method further comprises administering an immunosuppressive or immunomodulatory drug to modulate an immune response. In some embodiments, the immune response is a humoral response or antibody-mediated response.

[0277] In some embodiments, the implantation method prevents graft rejection or promotes graft survival.

[0278] The engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies including; but not limited to drugs which inhibit T-cell activation (e.g.; calcineurin inhibitors (CNI)); systemic immunosuppressants for universal transplant immunotolerance (corticosteroids such as methylprednisolone (MEDROL® or; SOLU-MEDROL®); prednisone or prednisolone); CNI such as tacrolimus (PROGRAF® or; ASTAFRAF®); cyclosporine (NEORAL®; SANDIMMUNE® or; GENGRAF®); co-stimulation blockade therapy such as Abatacept (ORENCIA®) and Belatacept (NULOJIX®); anti-metabolites such as Mycophenolate motefil (CELLCEPT® or; MYFORTIC®); Azathioprine (IMURAN®); mTORi such as Sirolimus (RAPAMUNE®); Everolimus (AFINITOR®); T-cell depleting monoclonal antibodies such as muromonab-CD3 (OKT3); Alemtuzumab (Campath® or; LEMTRADA®); ATG (THYMOBLOBULIN® or; ATGAM®); B-cell depleting monoclonal antibodies such as rituximab (RITUXAN®); proteasome inhibitors such as Bortezomib (VELCADE®); IL-2-Ra monoclonal antibodies such as daclizumab (ZENAPAX®); Basiliximab (SIMULECT®); lymphocyte integrin blockade monoclonal antibodies such as Natalizumab (TYSABRI®); N-Acetyl Cysteine (NAC); hepatitis B vaccine (HEPLISAV-B®); glecaprevir and pibrentasvir (MAVYRET®); sofosbuvir (VOSEVI®); obeticholic acid (OCALIVA®); elbasvir and grazoprevir (ZEPATIER®); cholic acid (CHOLBAM®); daclatasvir (DAKLINZA®); ombitasvir, paritaprevir, and ritonavir (TECHNIVIE™); simeprevir (OLYSIO™); sofosbuvir (SOVALDI®); telaprevir (INCIVEK™); boceprevir (VICTRELIS™); tenofovir disoproxil fumarate (VIREAD®); telbivudine (TYZEKA™); entecavir (BARACLUDE™); adefovir (HEPSERA®); peginterferon alfa-2a (PEGASYS®); peginterferon alfa-2b (PEGINTRON®); or ribavirin and twinrix. Additional agents include gliltazones and vitamin E.

[0279] In some embodiments, the engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies including but not limited to a PEGylated anti-CD28 monovalent monoclonal antibody fragment (e.g., anti-human CD28 FR104) or domain antibody such as lulizumab (BMS-931699), an IL-2Ra specific antibody for Treg expansion (e.g., a Fc IL-2 mutein (e.g., AMG-592)), a PEGylated IL-2 antibody, a humanized IgG 1 anti-CD40L antagonist (e.g., AT-1501 ), a bivalent anti-CD40L domain antibody such as letolizumab (BMS-986004), an Fc silent human IgG 1 anti-CD40 antibody such as VIB4920 or iscalimab (CFZ533), imlifidase, or a human anti-IL6 monoclonal antibody such as clazakizumab (CSL300).

[0280] In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least two weeks (e.g., at least three weeks, one month, two months, three months, four months, five months, six months, seven months, ten months, eleven months, one year, five years, ten years, or the lifetime of a patient in which the engineered tissue construct is implanted into). For example, in some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least three weeks. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least one month. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least two months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least three months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least four months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least five months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least six months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least seven months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least eight months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least nine months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least ten months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least eleven months. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least one year. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least five years. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for at least ten years. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists for the lifetime of a patient in which the engineered tissue construct is implanted into. In some embodiments, following implantation of the engineered tissue construct, the engineered tissue construct persists until an organ transplant occurs (e.g., until the patient receives a liver transplant).

[0281] In some embodiments, the hepatocytes or hepatocyte progenitor cells (e.g., hepatocytes e.g., primary human hepatocytes (PHH)) for an engineered tissue construct are obtained from a subject that has been diagnosed with a disorder (e.g., diabetes, a cardiovascular disease, a neurodegenerative disease, or a liver disease). In some embodiments, the subject is an adult or a pediatric patient (e.g., a human patient that is younger than 18 years old). The obtained hepatocytes or hepatocyte progenitor cells may be isolated and then expanded ex vivo under conditions that preserve or restore proliferative and functional capacity. In some embodiments, the hepatocytes obtained from the subject are reprogrammed at a time point prior to or after expansion to enhance hepatocyte function. In some embodiments, at a time point before or after expansion, the hepatocytes or hepatocyte progenitor cells are genetically modified (e.g., engineered) to secrete an incretin (e.g., an incretin listed in Table 1 ; e.g., an incretin that has the amino acid sequence of any one of SEQ ID NOs: 1 -11 ). In some embodiments, at a time point before or after expansion, the hepatocytes or hepatocyte progenitor cells are genetically modified to produce hypoimmunogenic cells. The hepatocytes or hepatocyte progenitor cells may be genetically modified to lack expression (e.g., have undetectable expression or less than 5% of a population hepatocytes or hepatocyte progenitor cells have detectable expression) of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, and PVR (e.g., one, two, three, four, five, or all six of the foregoing genes). The hepatocytes or hepatocyte progenitor cells may be genetically modified to express (e.g., overexpress) one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, CD64, and CD64t (e.g., one, two, three, four, five, six, seven, eight, nine, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, or all 38 of the foregoing genes). In some embodiments, the hepatocytes or hepatocyte progenitor cells are genetically modified to lack expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, and PVR (e.g., one, two, three, four, five, or all six of the foregoing genes) and also genetically modified to express (e.g., overexpress) one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, CD64, and CD64t (e.g., one, two, three, four, five, six, seven, eight, nine, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, or all 38 of the foregoing genes). The hepatocytes or hepatocyte progenitor cells (e.g., expanded and reprogrammed, engineered, and / or hypoimmunogenic hepatocytes or hepatocyte progenitor cells) may be encapsulated in an engineered tissue construct with a biocompatible scaffold and then administered to the same subject from which the hepatocytes or hepatocyte progenitor cells were obtained (i.e. , an autologous cell therapy) via any suitable route of administration, e.g., a route of administration described herein. The engineered tissue construct may be administered to the same subject as an autologous cell therapy to treat a disease or condition (e.g., obesity, diabetes, a cardiovascular disease, a neurodegenerative disease, or a liver disease) in the subject.

[0282] Examples

[0283] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0284] Example 1 : Engineered Tissue Constructs Containing Hypoimmunogenic Hepatocytes that Secrete GLP-1.

[0285] A population of cells that includes hypoimmunogenic hepatocytes with an exogenous polynucleotide that includes a promoter, a coding sequence for GLP-1 and a secretory signal are cultured with fibroblasts to form aggregates, e.g., as described in PCT Pub. No. WO 2023 / 076296. In some examples, the hypoimmunogenic hepatocytes are genetically engineered to lack expression of one or more of the following endogenous genes: B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and / or to express one or more of the following genes: CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD- L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG- 3, A2AR, BTLA, KIR, VISTA, CD64, CD64t, or any combination thereof.

[0286] In some examples, engineered tissue constructs are formed using the aggregates, e.g., as described in PCT Pub. No. WO 2023 / 076271 .

[0287] The aggregate or engineered tissue construct is administered or implanted into a human subject, e.g., as described in PCT Pub. No. WO 2023 / 076300.

[0288] It is expected that the methods described herein will result in the engineered hepatocytes secreting GLP-1 to impart a therapeutic effect on a subject.

[0289] Example 2: Detection of GLP-1 secreted by expanded human hepatocytes

[0290] Previous work was used to establish a cell engineering workflow to overexpress transgenes of interest in expanded human hepatocytes (eHH) through lentiviral delivery. Transduction experiments to date have included fluorescent markers for initial protocol development and the urea-cycle specific enzyme OTC to further cell potency work. However, the capability to overexpress transgenes in an expandable hepatocyte population has implications for treatment of new indications. Hepatocytes have several secretory organelles and extensive ER / Golgi networks which contribute to the role of hepatocytes as secretory workhorses. Glucagon-like peptide 1 (GLP-1 ) is a peptide hormone produced and secreted primarily by intestinal L-cells. GLP-1 , along with glucose-dependent insulinotropic polypeptide (GIP), are incretins, gut-derived peptide hormones that stimulate insulin secretion in a glucose-dependent manner. GLP-1 receptor agonist-based treatments have been associated with weight loss and a lower risk of hypoglycemia, two important considerations for the treatment of patients with type 2 diabetes.

[0291] In vivo, native GLP-1 is degraded rapidly by dipeptidyl peptidatse-4 (DPP-4) and renal clearance, resulting in a half-life of approximately 2 minutes. Due to this short turnover, only about 10-15% of GLP-1 reaches circulation intact. GLP-1 receptor agonists and DPP-4 inhibitors have been developed to increase GLP-1 activity. Structural modifications of GLP-1 , including the substitution of the amino acid alanine for glycine at position 8, has been shown to improve resistance to degradation by DPP-4. Both variants of GLP-1 , (7-37) and glycine 8 substitution, were considered in this experiment to explore the detection of biologically active GLP-1 secreted by eHH and understand how the structural modifications may improve protein half-life.

[0292] In this experiment, the established 17-day cell engineering workflow was used as an initial screening to determine if GLP-1 secreted by eHH could be detected. The goals of these experiments were to confirm that eHHs could be engineered to secrete GLP-1 (e.g., GLP-1 (7-37) and GLP-1 (7-37) A8G) and that the secretion products be detected via ELISA. Furthermore, these experiments also aimed to shows that eHH-secreted GLP-1 in culture media could activate GLP-1 receptors.

[0293] Experimental Overview

[0294] Transductions for this experiment followed a 17-day expansion + selection protocol. Briefly, cells were seeded at a density of 16k cells / well in a 6 well plate and expanded for 7 days. Following this, a 24 hr transduction and 48hr recovery period in EXPAND 3.0 10% KOSR Media (Table 2) was done before doing antibiotic selection using puromycin.

[0295] Table 2. Expand 3.0 Cocktail

[0296] Puromycin was added to the culture media for 7 days at a concentration of 4 pg / mL to remove untransduced cells. Supernatants were collected after transduction during each subsequent media exchange. A brief timeline of the experiment is shown in FIG. 1 .

[0297] Experimental groups included untransduced eHH, eHH transduced with GLP-1 (7-37), and cells transduced with DPP4 resistant GLP-1 (7-37) A8G. While the GLP-1 (1 -37) was identified from processing, the N-terminally truncated product, GLP-1 (7-37) was found to recognize the GLP-1 pancreatic receptor and is the active species in vivo. Given that this 7-37 sequence starts with histidine, a signal peptide sequence with a start codon followed by furin protease recognition site was included in the N-terminus of GLP-1 (7-37) in order to initiate translation and aid secretion from the cells. Following proteolytic cleavage by furin, GLP-1 (7-37) was the active secreted peptide. The construct included a human Ig kappa light chain V-lll region signal peptide, an SFFV constitutive promoter, and a puromycin selection marker. The construct design is shown in FIG. 2.

[0298] Untransduced eHH were seeded following the same protocol and expanded for 17 days. Cells were transduced at both MOI 5 and MOI 10 for this initial experiment to understand the appropriate viral dosage. Groups with and without puromycin selection were used to further the understanding of how antibiotic selection affects the efficiency of transduction and how puromycin specifically affects these engineered cells. The primary readout from this experiment was to perform GLP-1 ELISA with collected supernatants to confirm detection of the presence of secreted GLP-1 in culture media for both variants following standard cell engineering workflow. Detailed protocols for the experiments are shown below.

[0299] Methods

[0300] Thawing and Plating

[0301] 1 . Thaw 2 vials of laminin at 4°C. (Each vial has 1 ml of 100 pg / mL laminin and was thawed for about 10 to 20 minutes.

[0302] 2. Thaw Human Hepatocyte plating media supplement (HHPMS) at room temperature for about about 2- 3hours.

[0303] 3. Prepare plasmatherm by permitting to warm to 37 C and the allow to sit for an additional 15 minutes or longer.

[0304] 4. Place Gibco thaw media in Plasmatherm for 30 min before starting the thawing of cell stock

[0305] Coating-Laminin 521 (Thermofisher, A29249)

[0306] 1 . Label plates with experiment name, date, and initials.

[0307] 2. Coat 6wp flasks with Recombinant human Laminin 521 (rhLaminin-521 ) (40x, 1 :39) and allow coating for a 2 hour in normoxia incubator at 37 °C, 5% CO2)

[0308] • Stock Concentration 100 pg / mL

[0309] • Final coating was : 2.5 pg / mL, 0.5 pg per cm2. Each vial of laminin can prepare enough coating media for 30 wells of a 6wp Seeding Media Preparations

[0310] 1 . Make 265 ml of Human Hepatocyte Complete Plating Medium (HHCPM) by transferring 15 ml of HHPMS into 250 mL of Lifenet Human Hepatocyte Plating Media. Add 15 mL of HHPMS to the bottle of HHPM and transfer 2.65 mL of 100x Pen / Strep. Pass the plating media through a 500 mL 0.22 pm media filter to sterilize.

[0311] 2. Make one 50 pL aliquot of Trypan Blue for counting cells from the thawed vial for plating

[0312] 3. Add 30 pL of HHPM to each 50 pL Trypan Blue aliquot.

[0313] 4. Put 50 mL tube of sterile LNH Human Hepatocyte Thawing Medium in the plasmatherm 20 minutes prior to thawing the vial(s).

[0314] Thawing Cells for Recovery

[0315] Total Cell Demand: 480k cells

[0316] 1 . Make sure everything is ready in the BSC before retrieving vials including: serological pipettes, tips, thaw media, plating media, trypan blue and hemocytometers for counting. Additionally, make sure the thaw media is in the plasmatherm and warmed to 37 °C and immediately before placing the cryovials in the plasmatherm to thaw remove the thaw media from the plasmatherm and spray into BSC.

[0317] 2. Retrieve the vial of PHH from liquid nitrogen.

[0318] 3. Transfer 1 vial to the plasmatherm and permit to thaw for approximately 3-4 minutes. Watch the vials and when liquid start to flow into the cap remove them from the plasmatherm.

[0319] 4. Remove the cap from a single PHH cryovial and decant the full contents into a 50mL tube of PHH- warmed Thaw Media. Wash the PHH vial with 1 mL of the PHH Thaw Media using a 2 mL pipette. Dispense into the 50mL conical tube and gently invert three (3) times to ensure even distribution.

[0320] 5. Immediately centrifuge PHHs using the following parameters: a. Temperature: Ambient b. ACC: 9 c. DEC: 9 d. Speed: 10Oxg e. Time: 8 min

[0321] 6. Using a 2 mL aspiration pipet connected to the vacuum line, slowly aspirate supernatant.

[0322] 7. Resuspend the PHHs with 5 mL of pre-warmed plating media (HHPM).

[0323] 8. Take a 20 pL sample of the resuspended cells and dilute it 5x in 50 pL TB and 30 pL of media plating media (HHPM) for counting

[0324] 9. Determine the cell suspension density and create seeding suspensions for 16,000 cells per well

[0325] 10. Transfer flasks and plates to normoxia incubator.

[0326] 11 . After four hours aspirate plating media and add EXPAND 3.0 + 5% KOSR media to plates for expansion (25 mL). Move plates to hypoxia incubator.

[0327] Expansion and Media Preparations

[0328] Preparation of Expand media

[0329] Expand Basal media Expand complete media (includes growth factors and KOSR)

[0330] Supernatant Collection & Processing

[0331] • Collect 1 mL supernatant from each well into pre-labeled 1 .5mL tube

[0332] • Centrifuge supernatant samples (27) at 1500rpm (~400g) for 10 min at 4 °C

[0333] • Split supernatant into 400 mL aliquot for GLP-1 ELISA and 400 mL aliquot for GLP-1 activity assay in pre-labeled tubes

[0334] • Store purified supernatants at -80 °C in pre-labeled box

[0335] Media Exchange

[0336] • Aspirate the remaining 1 mL media from all wells, making sure to switch aspirator tips in between plates

[0337] • Prepare fresh EXPAND 3.0 10% KOSR media and EXPAND 3.0 10% KOSR + Puromycin media

[0338] • Exchange media as follows: o eHH untransduced and plates transduced with GLP-1 (7-37) or GLP-1 (7-37) A8G receive regular EXPAND 3.0 10% KOSR media o half with GLP-1 (7-37) or GLP-1 (7-37) A8G get EXPAND media + puromycin, other half get no puromycin

[0339] T ansduction

[0340] On day 7, plates were imaged to determine if the hepatocytes were ready for transduction. Prepare transduction media following protocol below in EXPAND 3.0 + 10% KOSR media.

[0341] Harvest three wells from "counting" plate and average the cell count to estimate total cell count for transduction calculations below.

[0342] For each MOI, add appropriate volumes of virus constructs to EXPAND 10% media.

[0343] Six Well Plate Harvest and Counting

[0344] 1 . Swish media in plate and tilt plate to collect media and dead cells at bottom then use the vacuum aspirator to aspirate media to remove dead cells and media.

[0345] 2. Add 2 mL of pre-warmed (37 °C) DPBS and swish to wash any remaining dead cells and media into suspension.

[0346] 3. Aspirate the DPBS from the wells and Add 1 .5 mL / well of pre-warmed (37 °C) accutase reagent keeping the culture surface level while adding the accutase.

[0347] 4. Place the plate in incubator and check at 4x magnification on the Zeiss microscope to determine if cells have lifted off the surface in clumps.

[0348] 5. Once the cells are detached from the plate surface (check by agitating plate under microscope and if the clump still moves with the liquid after agitating it is detached) bring the tubes of warmed Advanced DMEM / F12 and spray into the BSC.

[0349] 6. Agitate the dissociated cell suspension in the well and tilt the plate to collect the cell suspension

[0350] 7. Using a 5 mL pipette, triturate the accutase cell suspension 5 times to break up clumps to collect the cell suspension from the well and transfer suspension into the 15 mL conical tube containing 3.5mL of pre-warmed Advanced DMEM / F12

[0351] 8. Using the same 5 mL serological pipette forcefully pipette out 2.5 mL of pre-warmed Advanced DMEM / F12 onto the well surface from the other 15 mL tube to the well and swish around to collect any remaining cells. Do this wash 2 times.

[0352] 9. Tilt the plate and collect the rinse media and transfer to tube containing the bulk of the cell suspension.

[0353] 10. Centrifuge the cell collection tube at 200 g for 5 mins.

[0354] 11 . After centrifugation is complete, aspirate the supernatant from the pellet using the vacuum aspirator with a 1000 pL tip on the end and flick to dislodge the pellet.

[0355] 12. Resuspend the cell pellet in DMEM / F12 depending on the size of the cell pellet.

[0356] 13. Obtain cell counts.

[0357] Average Cell Count across three wells was 58,000 cells / well.

[0358] Use this for viral titre calculations below.

[0359] Reference Formula:

[0360] X pl Lentivirus = [# cells] * [MOI] * 1000 / [viral TU / mL]

[0361] 1 . Gently aspirate 2 mL EXPAND 3.0 + 5% KOSR media from each 6 wp well, be careful not to disturb adherent cells.

[0362] 2. Slowly dropwise add 2 mL transduction media (containing virus at final dilution) to the side of each respective 6wp well to coat the cells.

[0363] 3. Allow hepatocytes to be transduced for 24 hrs at 37 °C in hypoxia.

[0364] Antibiotic Selection

[0365] After transduction, aliquot the appropriate amount of EXPAND 3.0 + 10% KOSR media into a conical tube and add 4 pg / mL puromycin. Subsequent media changes will occur with media containing antibiotics.

[0366] Add 1 .4 mL of puromycin (1 OOpg / mL) to 33.6mL EXPAND 10% KOSR media and use STERIFLIP® vacuum tube top filter. Add 2 mL warmed puromycin-containing media to two groups (12 wells total).

[0367] Add 2 mL of warmed EXPAND 10% KOSR media (no puromycin) to untransduced other groups of wells (15 wells total).

[0368] Observe cells closely during the selection phase to monitor for cell death. Selection should typically occur for 7 days (3-4 media exchanges) to effectively kill WT cells and allow transduced cells to recover.

[0369] Harvest and Sample Preparation

[0370] Supernatants collected from media exchange days post-transduction were stored and prepared for GLP-1 ELISA readouts. Cells from all groups will be lysed in RLT Lysis Buffer to save for potential RT- qPCR analysis in the future. Preparation of Lysis Buffer:

[0371] 1 . Add p-mercaptoethanol to RLT Lysis Buffer at 1% (10 pL BME for 1 mL RLT Buff er).

[0372] 2. add 1 OpL p-mercaptoethanol for every 100 pL of RLT Buff er (1 OOpL in 10mL)

[0373] Preparation of Cell Lysates:

[0374] 1 . Aspirate media from all wells following supernatant collection.

[0375] 2. Add 350 pL of RLT Lysis Buff er directly to wells. Gently pipette up and down until cells are lysed. Transfer 350 pL lysed cells into pre-labeled tube and store at -80 °C.

[0376] Data Analysis

[0377] Supernatant ELISAs - GLP-1 concentration

[0378] GLP-1 ELISA was used to quantitatively assess secreted GLP-1 concentrations of the two constructs from the two transduced cell lines. ELISA data was used to interpolate efficiency data between MOI 5 and 10 conditions, as well as selected vs. unselected groups.

[0379] Results

[0380] As shown in FIG. 3, DPP-4 was shown to be expressed by primary human hepatocytes (PHH), expanded human hepatocytes (eHH), expanded and matured human hepatocytes (emhh), fetal liver, adult liver, and iPSC derived hepatocytes.

[0381] As shown in FIG. 4 (ELISA on day 16 of the cell engineering protocol), GLP-1 (7-37) was detected in all conditions (MOI of 5 or 10) at relevant levels. GLP-1 (7-37) A8G was detected at low levels. It is unclear if this is related to the kit detection or lower levels of cell secretion.

[0382] Future Experiments

[0383] Future experiments will be aimed to determine bioactivity of cell-secreted GLP-1 . Sigma's Glucagon Like Peptide-1 (Active) ELISA kit may be used for this experiment for its specificity to biologically active forms of GLP-1 such as the GLP-1 (7-37) variant used. It does not detect other forms of GLP-1 (1 -36 amide, 1 -37, 9-36 amide, or 9-37). A GLP-1 R reporter assay kit will be used as shown in FIG. 5. The kit includes engineered cells, which constitutively express GLP-1 Receptor and engineered luciferase reporter gene functionally linked to cAMP Response Element (CRE). Quantifying relative changes in luciferase enzyme activity in the reporter cells offers a surrogate measurement of induced changes in GLP-1 R activity. The kit is optimized for screening small molecules diluted in proprietary Compound Screening Media (CSM), and the supernatant will be tested.

[0384] Other Embodiments

[0385] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0386] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0387] Other embodiments are within the claims.

Claims

CLAIMS1 . An engineered hepatocyte or hepatocyte progenitor cell comprising a polynucleotide encoding an incretin, wherein the polynucleotide comprises:(a) a promoter sequence;(b) a secretory signal sequence; and(c) a coding sequence encoding the incretin linked to the promoter sequence and the secretory signal sequence.

2. The engineered hepatocyte or hepatocyte progenitor cell of claim 1 , wherein the polynucleotide is an episomal vector within the hepatocyte or hepatocyte progenitor cell.

3. The engineered hepatocyte or hepatocyte progenitor cell of claim 1 , wherein the polynucleotide is integrated into a genome of the hepatocyte or hepatocyte progenitor cell.

4. The engineered hepatocyte or hepatocyte progenitor cell of claim 3, wherein the polynucleotide is integrated into a safe harbor locus of the genome of the hepatocyte or hepatocyte progenitor cell.

5. The engineered hepatocyte or hepatocyte progenitor cell of claim 1 , wherein the incretin is a glucagon- like peptide-1 (GLP-1 ) receptor agonist (RA), a gastric inhibitory peptide (GIP) RA, or a glucagon RA.

6. The engineered hepatocyte or hepatocyte progenitor cell of claim 5, wherein the GLP-1 RA is GLP-1 , GLP-2, proglucagon, oxyntomodulin, exendin-4, or a biologically active fragment thereof.

7. The engineered hepatocyte or hepatocyte progenitor cell of claim 5, wherein the GLP-1 is GLP-1 (1 - 37), GLP-1 (7-36), or GLP-1 (7-37).

8. The engineered hepatocyte or hepatocyte progenitor cell of claim 5, wherein the GIP RA is GIP.

9. The engineered hepatocyte or hepatocyte progenitor cell of claim 5, wherein glucagon RA is glucagon.

10. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -9, wherein the incretin is fused to a half-life extending moiety.11 . The engineered hepatocyte or hepatocyte progenitor cell of claim 10, wherein the half-life extending moiety is a fragment crystallizable (Fc) domain, albumin, an albumin binding peptide, an albumin binding domain, a lipid binding moiety, a cholesterol binding moiety, a glycosylation site (e.g., an O-linked glycosylation site or N-linked glycosylation site), or an antibody or antigen-binding fragment thereof.

12. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -11 , wherein the secretory signal sequence is a human oncostatin M (OSM), vesicular stomatitis virus G protein (VSV-G), mouse immunoglobulin (Ig) kappa, mouse Ig heavy, osteonectin, SPARC [secreted protein, acidic, rich incysteine] (BM40), secrecon, human IgKVIll, mouse IgKVIll, cluster differentiation 33 (CD33), tissue plasminogen activator (tPA), human chymotrypsinogen, human trypsinogen-2, human interleukin 2 (IL-2), gaussia luciferase, human serum albumin (HSA), influenza haemagglutinin, human insulin, or silkworm fibroin light chain (LC) signal sequence.

13. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -12, wherein the secretory signal sequence is located at an N-terminus of the polypeptide.

14. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -13, wherein the promoter is an endogenous promoter.

15. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -13, wherein the promoter is an exogenous promoter.

16. The engineered hepatocyte or hepatocyte progenitor cell of claim 15, wherein the exogenous promoter is a constitutive promoter or an inducible promoter.

17. The engineered hepatocyte or hepatocyte progenitor cell of claim 16, wherein the constitutive promoter is a cytomegalovirus (CMV), a CMV early enhancer element, a first exon and a first intron of chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene (CAG), chicken beta actin (CBA), CBA hybrid (CBh) , human elongation factor-1 alpha (EF1 a), EF1 alpha short (EFS), spleen focusforming virus (SFFV), ubiquitin C (UBC), murine stem cell virus (MSCV), simian virus 40 (SV40), phosphoglycerate kinase (PGK), minimal cytomegalovirus promoter (minCMV), minimal herpes simplex virus thymidine kinase (minTK), or (ybTATA) promoter.

18. The engineered hepatocyte or hepatocyte progenitor cell of claim 16, wherein the inducible promoter is a chemically inducible promoter, a temperature inducible promoter, a light inducible promoter, or a physiological responsive promoter.

19. The engineered hepatocyte or hepatocyte progenitor cell of claim 18, wherein the chemically inducible promoter is a tetracycline responsive element (TRE), cymene repressor (CymR), cumate switch (SparQ), a lac operon (pLac) promoter, an arabinose inducible promoter, an Aspergillus nidulans alcA promoter, or a LexA promoter.

20. The engineered hepatocyte or hepatocyte progenitor cell of claim 18, wherein the temperature inducible promoter is an Hsp70 or Hsp90 derived promoter.21 . The engineered hepatocyte or hepatocyte progenitor cell of claim 18, wherein the light inducible promoter is a YFI or FixK2 promoter.

22. The engineered hepatocyte or hepatocyte progenitor cell of claim 18, wherein the physiologically responsive promoter, is an L-pyruvate kinase (LPK) promoter, glucose transporter-2 (GLUT2) promoter, carbohydrate response element binding protein (ChREBP) promoter, insulin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, glucose-6-phosphatase (G6Pase) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, activating transcription factor 6 (ATF6) promoter, FAS (TNFRSF6 / CD95 / APO-1 ) promoter, or insulin-like growth factor binding protein 1 (IGFBP- 1 ) promoter.

23. The engineered hepatocyte or hepatocyte progenitor cell of claim 16, wherein the inducible promoter is a synthetic transcription factor regulatable promoter.

24. The engineered hepatocyte or hepatocyte progenitor cell of claim 23, wherein the synthetic transcription factor is a zinc finger nuclease, a transcription activator-like effector nucleases (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 nuclease.

25. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -24, wherein the engineered hepatocyte or hepatocyte progenitor cell further comprises a second coding sequence encoding a second polypeptide.

26. The engineered hepatocyte or hepatocyte progenitor cell of claim 25, wherein the second polypeptide is a dipeptidyl peptidase IV (DPP-IV) inhibitor or an incretin.

27. The engineered hepatocyte or hepatocyte progenitor cell of claim 26, wherein the incretin is a GLP-1 RA, a GIP RA, or a glucagon RA.

28. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 25-27, wherein the polynucleotide encodes a linker between the coding sequence and the second coding sequence.

29. The engineered hepatocyte or hepatocyte progenitor cell of claim 28, wherein the linker comprises a cleavable peptide.

30. The engineered hepatocyte or hepatocyte progenitor cell of claim 29, wherein the cleavable peptide is a furin site or a 2A peptide.31 . The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 25-30, wherein the polynucleotide comprises an internal ribosomal entry site between the coding sequence and the second coding sequence.

32. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 25-31 , wherein the polynucleotide is polycistronic.

33. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -32, wherein the hepatocyte or hepatocyte progenitor cell is a hypoimmunogenic hepatocyte or hepatocyte progenitor cell.

34. The engineered hepatocyte or hepatocyte progenitor cell of claim 33, wherein the hypoimmunogenic hepatocyte or hepatocyte progenitor cell lacks expression of one or more of the following endogenous genes: beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or a combination thereof.

35. The engineered hepatocyte or hepatocyte progenitor cell of claim 34, wherein the hypoimmunogenic hepatocyte or hepatocyte progenitor cell comprises one or more alterations that inactivate the endogenous B2M, HLA-A, HLA-B, HLA-C, CIITA, or PVR gene.

36. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 33-35, wherein the hypoimmunogenic hepatocyte or hepatocyte progenitor cell expresses one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 2 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3- dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V- domain Ig suppressor of T cell activation (VISTA), CD64, truncated CD64 (CD64t), or a combination thereof.

37. The engineered hepatocyte or hepatocyte progenitor cell of claim 36, wherein the expression of the one or more genes is controlled by one or more inducible promoters or constitutive promoters.

38. The engineered hepatocyte or hepatocyte progenitor cell of claim 36 or 37, wherein the hypoimmunogenic cell overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL- 10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, CD64, CD64t, or a combination thereof relative to a wild-type hepatocyte or hepatocyte progenitor cell.

39. The engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -38, wherein the hepatocyte or hepatocyte progenitor cell is a pluripotent stem cell derived hepatocyte or hepatocyte progenitor cell or an expanded hepatocyte or hepatocyte progenitor cell, or an expanded and partially reprogrammed hepatocyte or hepatocyte progenitor cell.

40. An engineered tissue construct comprising a population of the hepatocytes or hepatocyte progenitor cells of any one of claims 1 -39 and a biocompatible scaffold.41 . The engineered tissue construct of claim 40, further comprising a second population of hepatocytes or hepatocyte progenitor cells, wherein the second population of hepatocytes or hepatocyte progenitor cells comprises a polynucleotide encoding a second polypeptide, wherein the polynucleotide comprises:(a) a promoter sequence;(b) a secretory signal sequence; and(c) a coding sequence encoding the second polypeptide operably linked to the promoter sequence and the secretory signal sequence.

42. The engineered tissue construct of claim 41 , wherein the polynucleotide encoding the second polypeptide is integrated in a genome of the second population of hepatocytes or hepatocyte progenitor cells.

43. The engineered tissue construct of claim 41 or 42, wherein the polynucleotide encoding the second polypeptide is integrated into a safe harbor locus of the genome of the second population of hepatocytes or hepatocyte progenitor cells.

44. The engineered tissue construct of any one of claims 40-43, wherein the biocompatible scaffold comprises fibrin, alginate, polyethylene glycol (PEG), modified PEG, agarose, chitosan, poly(lactic-co- glycolic acid) (PLGA), cellulose acetate, or a combination thereof.

45. The engineered tissue construct of claim 44, wherein the fibrin comprises human fibrin.

46. The engineered tissue construct of any one of claims 40-45, further comprising a population of stromal cells.

47. The engineered tissue construct of claim 46, wherein the stromal cells comprise fibroblasts.

48. The engineered tissue construct of claim 47, wherein the fibroblasts are normal human dermal fibroblasts.

49. The engineered tissue construct of any one of claims 46-48, wherein the population of stromal cells comprise a polynucleotide encoding an incretin.

50. The engineered tissue construct of any one of claims 46-49, wherein the population of stromal cells are hypoimmunogenic stromal cells.51 . A method of expressing an incretin in a human subject, the method comprising implanting the engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -35 or the engineered tissue construct of any one of claims 40-50 in the subject, thereby expressing the incretin in the human subject.

52. The method of claim 51 , wherein the method treats a disease or disorder in the subject.

53. The method of claim 52, wherein the disease or disorder is diabetes, obesity, a cardiovascular disease, a neurodegenerative disease, or steatohepatitis.

54. The method of claim 53, wherein the cardiovascular disease is high blood pressure, hypertension, arrhythmia, valve disease, coronary artery disease, heart failure, peripheral artery disease, aortic disease, pericardial disease, cerebrovascular disease, deep vein thrombosis, or a congenital heart disease.

55. The method of claim 53, wherein the neurodegenerative disease is dementia, Alzheimer’s disease, frontotemporal dementia, chronic traumatic encephalopathy, Lewy body dementia, multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, or prion disease.

56. The method of claim 53, wherein the steatohepatitis is non-alcoholic steatohepatitis.

57. A method of producing the engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -39, the method comprising packaging the polynucleotide in a viral vector and transducing the hepatocyte or hepatocyte progenitor cell with the viral vector.

58. The method of claim 57, wherein the viral vector is a lentiviral vector.

59. A method of producing the engineered hepatocyte or hepatocyte progenitor cell of any one of claims 1 -39, the method comprising contacting the hepatocyte or hepatocyte progenitor cell with the polynucleotide.

60. The method of claim 59, wherein the method comprises inserting the polynucleotide into a locus of the hepatocyte or hepatocyte progenitor cell.61 . The method of claim 60, further comprising introducing one or more single guide RNAs (sgRNA) into the hepatocyte or hepatocyte progenitor cell that specifically hybridizes to the locus.

62. The method of claim 60 or 61 , further comprising providing a nuclease that introduces a double stranded DNA break to create the insertion site.

63. The method of claim 62, wherein the nuclease is a Cas9 nuclease.

64. The method of claim 59, wherein the contacting comprises transduction, transformation, homologous recombination, base editing, prime editing, transposon mediated delivery, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, diethylaminoethyl (DEAE)-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct micro-injection, or nanoparticle-mediated nucleic acid delivery.

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