Pancreatic islet organoids as protein factories

Abstract

Provided are genetically modified pancreatic islet organoids that encode polypeptides that are not endogenously expressed in pancreatic islet cells and methods for their use to treat diseases and disorders associated with reduced or absent expression of the polypeptides.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 173,622, filed April 12, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Description Statement A computer readable form of the Sequence Listing has been filed electronically herewith and is incorporated by reference in its entirety into this application. The Sequence Listing is contained in a file with the filename "20-1719-WO-SeqList_ST25.txt" created on April 5, 2022, which is 3kb in size. [Background technology]

[0003] Pancreatic islets of Langerhans are micro-organs that form the endocrine portion of the pancreas. They consist of endocrine alpha, beta, delta, epsilon and PP cells, which produce and secrete glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide, respectively. Pancreatic islet organoids (also called pseudoislets), formed by self-assembly of islet cells, followed by isolation and genetic manipulation, behave similarly to native islets. Summary of the Invention

[0004] In one aspect, the present disclosure provides a genetically modified pancreatic islet organoid comprising a recombinant expression vector, the recombinant expression vector comprises (a) a nucleic acid sequence encoding a polypeptide, and (b) a suitable control sequence operably linked to the nucleic acid sequence.In one embodiment, the recombinant expression vector is present in a pancreatic islet organoid cell that does not endogenously express the polypeptide.In another embodiment, the polypeptide is not endogenously expressed in pancreatic islet cells.In further embodiments, the polypeptide is a therapeutic polypeptide, or its analog.In some embodiments, the therapeutic polypeptide is selected from the group consisting of a polypeptide hormone and an enzyme replacement protein.In other embodiments, the therapeutic polypeptide comprises a polypeptide hormone selected from the group consisting of thyroid stimulating hormone (TSH), prolactin, parathyroid hormone (PTH), leptin, follicle stimulating hormone (FSH), and growth hormone (GH), or their analog. In some embodiments, the therapeutic polypeptide comprises a polypeptide hormone selected from the group consisting of thyrotropin releasing hormone (TRH), renin, gastrin, vasoactive intestinal peptide (VIP), vasopressin (ADH), oxytocin (OXY), melanocyte stimulating hormone (MSH), calcitonin, cholecystokinin (CCK), atrial natriuretic peptide (ANP), angiotensin, amylin, and adrenocorticotropic hormone (ACTH), or analogs thereof.In other embodiments, the therapeutic polypeptide comprises an enzyme replacement protein, the enzyme replacement protein being selected from the group consisting of factor VII (eptacog alfa), factor VIII, factor IX, factor XIII (catridecacog alfa), von Willebrand factor, taliglucerase alfa, agalsidase alfa or beta, imiglucerase, velaglucerase alfa, alglucosidase alfa, galsulfase, dornase alfa, laronidase, conestat alfa (C1 esterase inhibitor), pegloticase alfa-1-proteinase inhibitor, asfotase alfa (Strensic), idursulfase, elosulfase alfa, valiase, selbelipase alfa, epoetin theta, The therapeutic agent is selected from the group consisting of teta (Eporatio), beta (NeoRecormon), zeta (Retacrit), darbepoetin alfa (Aranesp), luspatercept (Reblozyl), filgrastim, lenograstim, and von Willebrand factor-cleaving protease, or an analog thereof.In various embodiments, the therapeutic polypeptide is an enkephalin, endorphin, substance P, neurotensin, neuropeptide-Y, bombesin, brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), neuotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cholinergic differentiation factor, cardiotrophin-1, oncostatin M, tumor necrosis factor (TNF), Neu differentiation factor, heregulin, acetylcholine receptor-inducing activity, glial cell growth factor (GGF), glial cell line-derived neurotrophic factor (GDNF), artemin, neurturin, persephin, bone morphogenetic protein-1 (OP-1), bone morphogenetic protein (BMP), growth differentiation factor, ephrin, epidermal growth factor (EGF), insulin-like growth factor (IGF), blood plasma membrane protein (BPMP), or a combination thereof. and neurotrophic factors selected from the group consisting of platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), granulocyte colony stimulating factor (G-CSF), serine protease inhibitors, protease nexin-1, the hedgehog family of inducible proteins, agrin, laminin 2, neuroimmunophilin, pigment epithelium derived factor (PEDF), activity-dependent neuroprotective protein (ADNP), neuritin (activity-induced neurotrophic factor), angiogenic growth factor, brain dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), peptide-6, davunetide (derived from ADNP), and cerebrolysin.

[0005] In some embodiments, the recombinant expression vector comprises a viral vector, including, but not limited to, a retroviral vector, a lentiviral vector, a murine leukemia virus (MMLV) vector, a murine stem cell virus (MSCV) vector, an adenoviral vector, a herpes simplex virus vector, a baculovirus vector, and an adeno-associated virus vector. In other embodiments, the expression vector comprises a non-viral vector.

[0006] In some embodiments, the suitable regulatory sequence is pancreatic tissue specific and is selected from the group consisting of midkine (MK) promoter, cyclooxygenase-2 (Cox2)M promoter, Cox 2L promoter, vascular endothelial growth factor (VEGF) promoter, caveolin 1 promoter, fms-like receptor tyrosine kinase 1 (FLT-1) promoter, sloppy paired 1 (SLP-1) promoter, gastrin releasing peptide (GRP) promoter, epithelial glycoprotein 2 (EGP-2) promoter, insulin promoter, and glucagon promoter. In other embodiments, the recombinant expression vector comprises an adeno-associated virus vector and the insulin promoter is RIP1 or RIP2. In further embodiments, the suitable regulatory sequence is selected from the group consisting of a cytomegalovirus (CMV) promoter, a chicken β-actin (CAG) promoter, a Rous sarcoma virus (RSV), a simian virus 40 (SV40), a mammalian elongation factor 1 alpha (EF1α) promoter, a muscle creatine kinase (MCK) promoter, a human phosphoglycerate kinase 1 (PGK1) promoter, and a tetracycline response element (TRE)-tight promoter.

[0007] In another embodiment, the present disclosure provides a composition comprising any embodiment or combination of embodiments herein genetically modified pancreatic islet organoid and silk matrix.In one embodiment, silk is spider silk.In another embodiment, silk is functionalized with cell binding motif.

[0008] In another aspect, the present disclosure provides the use and method for treating disorder, comprising administering the genetically modified pancreatic islet organoid of any embodiment or combination of embodiments herein, or its composition, to treat disorder.In one embodiment, genetically modified pancreatic islet organoid or composition is transplanted into the eye, pituitary gland, pancreas, small intestine, stomach, brain, kidney, parathyroid gland, duodenum, thyroid gland, liver, heart, ovary, testis, fat, and / or skin of the subject in need thereof.In another embodiment, the nucleic acid sequence encodes the polypeptide listed in the left hand column of table 1, and the subject has the disorder listed in the right hand column of table 1 in the same row as the polypeptide.

[0009] In another aspect, the present disclosure provides a process for producing genetically modified islet organoids comprising islet cells, comprising: (a) introducing a recombinant expression vector into islet cells, comprising (i) a nucleic acid sequence encoding a polypeptide that is not endogenously expressed in islet cells, and (ii) a suitable control sequence operably linked to the nucleic acid sequence; and (b) culturing the islet cells comprising the recombinant expression vector in vitro to form genetically modified islet organoids comprising islet cells.In one embodiment, the introducing step (a) further comprises: (a1) preparing an aqueous mixture of islet cells comprising recombinant expression vectors using silk protein that can be assembled into water-insoluble macrostructures, and optionally further comprises laminin; (a2) allowing silk protein to assemble into water-insoluble macrostructures in the presence of islet cells comprising recombinant expression vectors, thereby forming a 3D silk matrix of islet cells comprising recombinant expression vectors; culturing step (b) comprises culturing the islet cells comprising recombinant expression vectors in vitro in silk matrix to form genetically modified islet organoids comprising islet cells. In another embodiment, the process further comprises: (c) disposing the genetically modified islet organoids comprising islet cells on a 3D silk matrix consisting of a water-insoluble macrostructure of silk protein, and optionally further comprising laminin; and (d) allowing the genetically modified islet organoids comprising islet cells to attach to the 3D silk matrix. [Brief description of the drawings]

[0010] [Figure 1a-1c]Figure 1a-c shows the in vitro characterization of leptin-expressing pancreatic islet organoids. (a) Schematic of vAd-RIP-leptin-OFF. The rat insulin-1 promoter (rIns-1) drives the expression of the synthetic transcription factor rTA (Tet-off) and the green fluorescent protein ZsGreen™ in pancreatic beta cells. The TRE-tight promoter drives the expression of mouse leptin and the red fluorescent protein mCherry™. The two expression cassettes are separated by a transcription blocker sequence (TB). Binding of rTA to the TRE-tight promoter induces the expression of leptin and mCherry™ in the absence of doxycycline, whereas the addition of doxycycline stops the expression of the two proteins. The IRES element between the elements ensures the stoichiometric expression of the two proteins under the same promoter. (b) Representative maximum projection of confocal imaging of 3D stacks of leptin-expressing islet organoids cultured in vitro for 4 weeks (first experimental set) showing reflection, ZsGreen™, mCherry™ expression and their overlay. Scale bar: 50 μm. (c) Representative maximum projection of confocal imaging of 3D stacks of leptin-expressing islet organoids cultured in vitro for 4 weeks (second experimental set) showing reflection, ZsGreen™, mCherry™ expression and their overlay. Scale bar: 50 μm. [Figure 2a-2g]Figure 2a-g show the in vitro characterization of leptin-expressing islet organoids from the first set of experiments. (a) Photograph of an eye containing a leptin-expressing islet organoid graft (red arrow) 9 weeks after transplantation. (b) Maximum projection of a 3D stack of a leptin-expressing islet organoid graft (red arrow) obtained by confocal imaging 9 weeks after transplantation; (ba) reflection, (bb) mCherry™ fluorescence, (bc) ZsGreen™ fluorescence, (bd) overlay images; scale bar 500 μm. (c) Body weight of transplanted and control mice over the experimental period. (d) Area under the curve (AUC) for ipGTT of transplanted and control mice over the experimental period. (e) Fasting blood glucose of transplanted and control mice over the experimental period. (f) Plasma insulin levels of transplanted and control mice at the end of the experiment. (g) Plasma C-peptide levels of transplanted and control mice at the end of the experiment. (d, e) *p<0.05. (c~g)n=3. [Figures 3a-3g] Figure 3a-g show the in vivo characterization of leptin-expressing islet organoids from the second experimental set. (a) Photograph of an eye containing a leptin-expressing islet organoid graft (red arrow) 6 weeks after transplantation. (b) Maximum projection of a 3D stack of a leptin-expressing islet organoid graft (red arrow) obtained by confocal imaging 12 weeks after transplantation; (ba) reflection, (bb) mCherry™ fluorescence, (bc) ZsGreen™ fluorescence, (bd) overlay images; scale bar 300 μm. (c) Body weight, (d) area under the curve (AUC) for ipGTT, (e) fasting blood glucose, (f) plasma insulin levels and (g) plasma C-peptide levels of transplanted and control mice during the experimental period. (c-g) *p<0.05, **p<0.01, ***p<0.001; n=7 for transplanted group and 5 for control group. [Figure 4a-4d]Figure 4a-d show characterization of leptin-expressing islet organoid grafts from animals treated with doxycycline and removed at the end of the experiment. (a) Fluorescence microscopy images of leptin-expressing islet organoid grafts from transplanted non-doxycycline treated animals; (aa) DAPI staining; (ab) ZsGreen™ fluorescence; (ac) mCherry™ fluorescence; (ad) overlay. (b) Fluorescence microscopy images of leptin-expressing islet organoid grafts from transplanted doxycycline treated animals; (ba) DAPI staining, (bb) ZsGreen™ fluorescence, (bc) mCherry™ fluorescence, (bd) overlay. (c) Immunohistochemistry images of leptin-expressing islet organoid grafts from transplanted non-doxycycline treated animals; (ca) DAPI staining, (cb) C-peptide staining, (cc) leptin staining, (cd) overlay. (d) Immunohistochemistry images of leptin-expressing islet organoid grafts from transplanted doxycycline-treated animals; (da) DAPI staining, (db) C-peptide staining, (dc) leptin staining, (dd) overlay. Scale bars for (a-d) are 50 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] All references mentioned are incorporated herein by reference in their entirety. In this application, unless otherwise indicated, the techniques used are those described in: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press These compounds can be found in any of a number of well-known references, such as the 1998 Catalog of Amplification Compounds (Ambion, Austin, TX), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0012] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0013] All embodiments of any aspect of this disclosure can be used in combination unless the context clearly dictates otherwise.

[0014] In a first aspect, the present disclosure provides a genetically modified pancreatic islet organoid comprising a recombinant expression vector, the recombinant expression vector comprises (a) a nucleic acid sequence encoding a polypeptide, and (b) a suitable control sequence operably linked to the nucleic acid sequence.

[0015] The present disclosure provides the novel and surprising finding that pancreatic islet organoids can be genetically modified to express any polypeptide, including non-pancreatic polypeptides (defined as polypeptides not normally produced in pancreatic islets), and that these genetically modified pancreatic islet organoids can be transplanted into subjects and used as protein factories for long-term treatment of diseases treatable using polypeptides produced by genetically modified pancreatic islet organoids. Genetically modified pancreatic islet organoids mimic miniature organs. Genetically modified pancreatic islet organoids have a structure and functionality well suited to excrete polypeptides into the bloodstream of a subject, and this functionality can be maintained over long periods of time compared to surgical transplantation of individual cell types. Pancreatic islet organoids can be transplanted into or adjacent to other organs, effectively becoming a "complete organ within another organ". This is a unique feature of genetically modified pancreatic islet organoids.

[0016] The therapeutic effect of the polypeptide can be further adjusted to enhance local effect by selecting the specific implantation site of the implant according to the disease.The compositions and methods disclosed herein are exemplified in a non-limiting manner by the creation of islet organoids genetically modified to express leptin.Leptin is a polypeptide that is not normally produced by islets.The leptin-producing genetically modified islet organoids are then successfully used to rescue the metabolic phenotype of ob / ob (leptin-deficient knockout) mice, as shown in the examples.

[0017] As used herein, "genetically modified pancreatic islet organoid" refers to a cell cluster generated in vitro that mimics the structure and function (insulin glucagon, amylin, somatostatin, ghrelin and pancreatic polypeptide secretion) of the pancreatic islet of Langerhans, and the cell cluster comprises at least pancreatic beta cells, but can also comprise pancreatic alpha cells, pancreatic polypeptide (PP) cells, epsilon cells and delta cells.In various embodiments, the genetically modified pancreatic islet organoid comprises at least beta cells and alpha cells; in other embodiments, the genetically modified pancreatic islet organoid comprises at least one, two or all three of pancreatic beta cells, alpha cells, and PP cells, epsilon cells and delta cells.

[0018] Pancreatic islets of Langerhans are multicellular microorgans that form the endocrine portion of the pancreas. Pancreatic islets contain perforated blood vessels, allowing efficient exchange of blood-borne factors that can stimulate the islets, resulting in the release of polypeptide hormones produced by the islets into the general blood circulation.

[0019] In one embodiment, pancreatic islet organoid cells can be genetically modified from pancreatic islets obtained from any animal with pancreatic islets suitable for use, including but not limited to any mammal, such as humans. In other embodiments, genetically modified pancreatic islet organoids can be generated from induced pluripotent stem cells or stem cells from any source using any suitable technique, including but not limited to those disclosed in US Patent Publication No. 20190211310, the entirety of which is incorporated by reference. In one non-limiting embodiment, genetically modified pancreatic islet organoids can be generated by culturing induced pluripotent stem cell (iPSC)-derived or human embryo-derived or cadaveric or human-derived) beta-like cells, and optionally structural components, in a three-dimensional matrix. In various embodiments, pancreatic islet organoids can be generated by culturing iPSC-derived (or human embryo-derived or cadaveric or human-derived) beta-like cells with adipose-derived stem cells and / or endothelial cells. In various embodiments, pancreatic islet organoids can be generated by culturing iPSC-derived (or human embryo-derived or cadaveric or human-derived) beta-like cells with iPSC-derived (or human embryo-derived or cadaveric or human-derived) alpha-like cells, iPSC-derived (or human embryo-derived or cadaveric or human-derived) delta-like cells, iPSC-derived (or human embryo-derived or cadaveric or human-derived) epsilon cells, iPSC-derived (or human embryo-derived or cadaveric or human-derived) PP cells, and / or iPSC-derived (or human embryo-derived or cadaveric or human-derived) duct-like cells.

[0020] In various non-limiting embodiments, the genetically modified pancreatic islet organoid of the present disclosure can comprise any one or more of the following cell types: iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) beta-like cell, iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) alpha-like cell, iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) delta-like cell, iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) PP cell, iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) epsilon cell, and iPSC-derived (or human embryo-derived or cadaver-derived or human-derived) duct-like cell.In some embodiments, the iPSC is human iPSC (hiPSC).In some embodiments, the stem cell comprises human embryonic stem cell (hESC).In some embodiments, the pancreatic islet organoid comprises adipose-derived stem cell and / or endothelial cell.

[0021] Any cell in genetically modified pancreatic islet organoid can be genetically modified to contain recombinant expression vector, including but not limited to alpha cell, beta cell, delta cell, PP cell, epsilon cell or duct-like cell.In one embodiment, beta cell contains recombinant expression vector and expresses the polypeptide that is not endogenously expressed by beta cell and / or non-pancreatic polypeptide.

[0022] Recombinant expression vector, including virus and non-virus vector, can be any vector suitable for expression in pancreatic islet organoid.Suitable virus vector includes but is not limited to retrovirus vector, lentivirus vector, murine leukemia virus (MMLV) vector, murine stem cell virus (MSCV) vector, adenovirus vector, herpes simplex virus vector, baculovirus vector and adeno-associated virus vector.In one non-limiting example, recombinant expression vector is adenovirus vector.

[0023] Any suitable nucleic acid sequence encoding a polypeptide for expression in pancreatic islets may be used. As used herein, the term "nucleic acid sequence" refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs.

[0024] In some embodiments, the nucleic acid sequence encoding the polypeptide may be modified from the known coding sequence, for example, by alteration in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in pancreatic islet organoids; alteration in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and / or addition; removal of one or more introns; insertion of one or more heterologous introns; deletion of one or more upstream or downstream control regions; insertion of one or more heterologous upstream or downstream control regions; deletion of 5' and / or 3' untranslated regions; insertion of heterologous 5' and / or 3' untranslated regions; and modification of polyadenylation sites.

[0025] As used throughout this application, the term "polypeptide" is used broadly to refer to a sequence of subunit amino acids of any length. Polypeptides of the invention may contain L-amino acids, D-amino acids (which are resistant to L-amino acid specific proteases in vivo), or a combination of D- and L-amino acids.

[0026] The recombinant expression vector comprises a suitable control sequence operably linked to the nucleic acid sequence encoding the polypeptide. The control sequence can be any control sequence that can affect the expression of the nucleic acid sequence encoding the polypeptide. The control sequence can be either pancreatic tissue-specific, pancreatic islet cell-specific (i.e.: beta cell-specific; alpha cell-specific; delta cell-specific; etc.) or non-pancreatic tissue-specific (i.e., general control sequence). Non-limiting examples of pancreatic tissue-specific regulatory sequences include the midkine (MK) promoter, Cox2M promoter, Cox 2L promoter, vascular endothelial growth factor (VEGF) promoter, caveolin 1 promoter, fms-like receptor tyrosine kinase 1 (FLT-1) promoter, sloppy paired-1 (SLP-1) promoter, gastrin releasing peptide (GRP) promoter, epithelial glycoprotein 2 (EGP-2) promoter, rat insulin promoter (RIP1 or RIP2) (beta cell specific), insulin promoter (beta cell specific), glucagon promoter (alpha cell specific), and somatostatin promoter (delta cell specific). Non-limiting examples of non-tissue specific regulatory sequences include cytomegalovirus (CMV) promoter, chicken β-actin (CAG) promoter, Rous sarcoma virus (RSV), Simian virus 40 (SV40), mammalian elongation factor 1 alpha (EF1α) promoter, muscle creatine kinase (MCK) promoter, human phosphoglycerate kinase 1 (PGK1) promoter, and tetracycline response element (TRE)-tight promoter. In one embodiment, the promoter can be an inducible promoter such as Tet-ON / OFF promoter, pLac promoter, or pBad promoter. In one non-limiting example, the regulatory sequence includes a pTRE-tight promoter. In one embodiment, the promoter can be a synthetic promoter that includes the transcriptional regulators of the promoters described above.

[0027] The expression vector of the present disclosure may include any other components suitable for use with the vector. The control sequence does not need to be adjacent to the nucleic acid sequence, so long as it functions to direct its expression. Thus, for example, an intervening untranslated but transcribed sequence can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered to be "operably linked" to the nucleic acid. The expression vector may include other control sequences, including but not limited to polyadenylation signals, termination signals, and ribosome binding sites. The control sequences used to promote the expression of the nucleic acid sequence of the present disclosure may be constitutive or inducible (promoted by any of a number of inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsive). In one embodiment, the expression vector in the pancreatic islet organoid is replicable by integration into the host chromosomal DNA.

[0028] In further embodiments, a nucleic acid sequence encoding a polypeptide, or an expression promoter, may be introduced into a pancreatic islet cell using the CRISPR / Cas9 system, other nucleases such as zinc finger nucleases or transcription activator-like effector nucleases (TALENs), or other suitable gene editing systems.

[0029] In one embodiment of the first aspect, the recombinant expression vector of genetically modified pancreatic islet organoid is present in the pancreatic islet organoid cell that does not endogenously express polypeptide.In this embodiment, recombinant expression vector is only present in the pancreatic cell type that does not endogenously express polypeptide; this allows the cell that contains recombinant expression vector to produce the polypeptide that the cell does not normally produce.

[0030] In one non-limiting example, genetically modified pancreatic islet organoid comprises beta cells comprising recombinant expression vectors comprising nucleic acids encoding polypeptides that are not endogenously expressed by pancreatic beta cells, but may be endogenously expressed by other pancreatic islet cells.In a further example, genetically modified pancreatic islet organoid comprises alpha cells comprising recombinant expression vectors comprising nucleic acids encoding polypeptides that are not endogenously expressed by alpha cells, but may be endogenously expressed by other pancreatic islet cells.In a further example, genetically modified pancreatic islet organoid comprises delta cells comprising recombinant expression vectors comprising nucleic acids encoding polypeptides that are not endogenously expressed by delta cells, but may be endogenously expressed by other pancreatic islet cells.Examples of pancreatic polypeptides that are differentially expressed in different pancreatic cell types include, but are not limited to, glucagon, insulin, amylin, glucagon-like peptide-1 (GLP-1), somatostatin, and vasoactive intestinal peptide (VIP). This embodiment may, for example, allow genetically modified islet organoids to produce a polypeptide that is usually produced in very low concentrations in islet cells at higher concentrations, and / or allow expression in islet cell types that allow secretion of the polypeptide under certain conditions.In one example, genetically modified islet organoids can be genetically modified to express VIP, which is mainly produced in the intestine, but can be produced in low concentrations in islet cells.Therefore, in this embodiment, genetically modified islet organoids that express VIP can be used to increase the concentration of VIP expressed by islet cells.In another non-limiting embodiment, a recombinant expression vector that can express GLP-1 is present in the islet beta cells of genetically modified islet organoids; the expression and secretion of GLP-1 allows rapid local delivery of GLP-1 to beta cells in response to high glucose conditions, thus providing improved diabetes treatment.In various exemplary embodiments, the recombinant expression vector comprises an adenovirus or adeno-associated virus vector, and suitable control sequences include an insulin promoter, including, but not limited to, a CMV promoter or a functional portion thereof capable of directing gene expression, the human insulin promoter, and insulin promoters such as RIP1 and RIP2.

[0031] In non-limiting examples, the encoded polypeptide can include synthetic (non-naturally occurring) polypeptides, polypeptide fragments, non-human polypeptides, and natural or synthetic polypeptide analogs.

[0032] In a further embodiment of the first aspect, the polypeptide expressed by genetically modified pancreatic islet organoid is not endogenously expressed in any pancreatic islet cell type.In this embodiment, the encoded polypeptide is not endogenously expressed by non-genetically modified pancreatic islet cell.

[0033] In all of these embodiments, the polypeptide may be a therapeutic polypeptide. Examples of such therapeutic polypeptides include, but are not limited to, polypeptide hormones or enzyme replacement proteins.

[0034] In one embodiment, the therapeutic polypeptide comprises a polypeptide hormone that is not endogenously expressed in pancreatic islets. Non-limiting examples of polypeptide hormones that are not endogenously expressed in pancreatic islets include parathyroid hormone (PTH), leptin, follicle stimulating hormone (FSH), growth hormone (GH), thyrotropin releasing hormone (TRH), renin, gastrin, vasopressin (ADH), oxytocin (OXY), melanocyte stimulating hormone (MSH), calcitonin, cholecystokinin (CCK), atrial natriuretic peptide (ANP), angiotensin, leutinizing hormone releasing hormone (LHRH), and adrenocorticotropic hormone (ACTH). In embodiments in which the polypeptide hormone is processed after expression, the nucleic acid may express the mature form or pre-(pro)protein version of the polypeptide hormone. As used herein, the mature form of the polypeptide hormone is the form of the polypeptide hormone after final processing. In one embodiment, the nucleic acid encodes the mature form of the polypeptide hormone.

[0035] In another embodiment, the therapeutic polypeptide comprises an incretin or enzyme replacement protein. The nucleic acid may encode any such incretin, including, but not limited to, glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), glucose-dependent insulinotrophic peptide (GIP), GLP-1 receptor binding polypeptide, GIP receptor binding polypeptide, glucagon receptor binding polypeptide, and double or triple complexes of glucagon, GIP, and glucagon receptor binding polypeptide. Nucleic acids include factor VII (eptacog alfa), factor VIII, factor IX, factor XIII (catridecacog alfa), von Willebrand factor, taliglucerase alfa, agalsidase alfa or beta, imiglucerase, velaglucerase alfa, alglucosidase alfa, galsulfase, dornase alfa, laronidase, conestat alfa (C1 esterase inhibitor), pegloticase alfa-1-proteinase inhibitor, asfotase alfa (Strensic), idursulfase, elosulfase alfa, valiase, selbelipase alfa, epoetin theta, The enzyme may encode any such enzyme replacement protein, including, but not limited to, teta (Eporatio), beta (NeoRecormon), zeta (Retacrit), darbepoetin alfa (Aranesp), luspatercept (Reblozyl), filgrastim, lenograstim, and von Willebrand factor-cleaving protease.

[0036] In further embodiments, the therapeutic polypeptide can further comprise neuropeptides and neurotrophic factors that are not endogenously expressed in pancreatic islets. The nucleic acids can include those encoding enkephalins, endorphins, substance P, neurotensin, neuropeptide-Y, bombesin, brain-derived neurotrophic factor, Neurotrophin-3 (BDNF), neurotrophin-4 (NGF), neuotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cholinergic differentiation factor, cardiotrophin-1, oncostatin M, growth promoting activity factor, tumor necrosis factor (TNF), Neu differentiation factor, heregulin, acetylcholine receptor-inducing activity, glial cell growth factor (GGF), glial cell line-derived neurotrophic factor (GDNF), artemin, neurturin, persephin, bone morphogenetic protein-1 (OP-1), bone morphogenetic protein (BMP), growth differentiation factor, ephrin, epidermal growth factor (EGF), transforming growth factor (TGFα and TGFβ), insulin-like growth factor (IGF), platelet-derived growth factor (PDG F), hepatocyte growth factor (HGF), granulocyte colony stimulating factor (G-CSF), serine protease inhibitor, protease nexin-1, hedgehog family of inducible proteins, agrin, laminin 2, ACh-induced activity (ARIA), neuroimmunophilin, pigment epithelium-derived factor (PEDF), activity-dependent neuroprotective protein (ADNP), neuritin (activity-induced neurotrophic factor), angiogenic growth factors, vascular endothelial growth factor (VEGF), brain dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), peptide-6, davunetide (derived from ADNP), and cerebrolysin.

[0037] It will be understood that genetically modified pancreatic islet organoid may comprise multiple recombinant expression vectors that can express the same or different polypeptides.In various embodiments, genetically modified pancreatic islet organoid may comprise two, three or more different recombinant expression vectors, each of which can express the same or different polypeptides.Different recombinant expression vectors may all be present in the same cell type in the organoid, or may be present in different cell types in the organoid.In one non-limiting embodiment, organoid may comprise two or more different expression vectors, all of which express the same polypeptide, but each of which is under the control of different promoters or other suitable regulatory elements, so that different expression vectors express the polypeptide only under different conditions and / or when present in the organoid cell type in which the promoter and / or regulatory element is effective. In another non-limiting embodiment, organoid may comprise two or more different expression vectors, all expressing different polypeptides, and each different polypeptide may be under the control of the same or different promoter or other suitable regulatory elements, so that different expression vectors may express different polypeptides under the same conditions (when using the same promoter and / or regulatory element), under different conditions (when using different promoter and / or regulatory element), and / or only when present in the organoid cell type for which the promoter and / or regulatory element is effective.In one particular embodiment, organoid may comprise two or three of the following: (1) a first expression vector, whose expression is under the control of insulin promoter, restricting expression to beta cells; (2) a second expression vector, whose expression is under the control of glucagon promoter, restricting expression to alpha cells; and (3) a third expression vector, whose expression is under the control of somatostatin promoter, restricting expression to delta cells.In other examples, the organoids may contain one or more expression vectors, expression of which is under the control of the CMV promoter and / or the rat insulin promoter, RIP1 or RIP2.

[0038] In a second aspect, the present disclosure provides a composition comprising any embodiment or combination of embodiments disclosed herein of genetically modified pancreatic islet organoid and silk matrix.Genetic modified pancreatic islet organoid may be attached on silk matrix or integrated into silk matrix.Silk matrix may have the shape of fiber, foam, film, fiber mesh, capsule, net, or gel, preferably fiber or foam.Silk matrix may comprise silk protein.Silk protein is preferably fibroin, such as silkworm fibroin, or spider silk protein.

[0039] Spider silk is a biocompatible material and can be produced through recombinant DNA technology. Spider silk can function as a cell scaffolding material for the culture of eukaryotic cells. Polymers of silk protein can be used by cells as cell scaffolds. Spiders have up to seven different glands that produce a wide variety of silk types with different mechanical properties and functions, including but not limited to dragline silk.

[0040] Spider silk proteins can form macrostructures, including films and bubbles, with attached and / or integrated cells, such as those of pancreatic islet organoids, and thus provide an internal 3D support for genetically modified pancreatic islet organoid cells. These macrostructures confer high seeding efficiency and result in rapidly engrafted pancreatic islet organoids. Compared to cultures on other cell scaffolds, cells in spider silk achieve a more tissue-like spread when integrated into the silk scaffold. This may improve the functionality and viability of transplanted pancreatic islet organoids.

[0041] Functionalized silk is a bioactive silk-based material with enhanced functionality for medical and biotechnology related applications. As used herein, "functionalized spider silk" refers to recombinant spider silk that contains cell-binding motifs and is modified to covalently bind functional groups or domains. Examples of functionalized spider silk and methods for producing and using same include, but are not limited to, those disclosed in International Publication No. 2007078239, US Patent Publication No. 20160024464, US Patent No. 10316069, and US Patent Publication No. 20190144819, which are incorporated by reference in their entirety. In certain preferred embodiments of these and other aspects, the silk protein is selected from the group consisting of RGD, IKVAV (SEQ ID NO:1), YIGSR (SEQ ID NO:2), EPDIM (SEQ ID NO:3), NKDIL (SEQ ID NO:4), GRKRK (SEQ ID NO:5), KYGAASIKVAVSADR (SEQ ID NO:6), NGEPRGDTYRAY (SEQ ID NO:7), PQVTRGDVFTM (SEQ ID NO:8), AVTGRGDSPASS (SEQ ID NO:9), TGRGDSPA (SEQ ID NO:10), CTGRGDSPAC (SEQ ID NO:11), and FN. cc From; preferably FN cc , GRKRK, IKVAV, RGD and CTGRGDSPAC, more preferably FN cc and CTGRGDSPAC, wherein FN cc is C 1 X 1 X 2 RGDX 3 X 4 X 5 C 2 and each X 1 , X 2 , X 3 , X 4 and X 5 are independently selected from naturally occurring amino acid residues other than cysteine; 1 and C 2 are connected via disulfide bonds. A preferred group of cell-binding motifs are FN cc, GRKRK, IKVAV, and RGD, especially FN cc , for example, CTGRGDSPAC.

[0042] The functionalized spider silk may include additional functional properties, such as electrical conductance, cell binding ability, growth factor bioactivity, molecular affinity, antimicrobial properties, and enzymatic activity. Such additional functional properties may be added, for example, by dip-coating, genetic engineering, and / or enzymatic coupling of the functionalized spider silk. (Examples of silk protein properties from patent WO2017137611.) In some embodiments, the functionalized spider silk may include immunosuppressive proteins such as PD-L1 or other immunomodulators to protect the genetically modified pancreatic organoids from immune responses. The functionalized spider silk may also include factors that enhance angiogenesis or other growth factors, such as fibronectin, VEGF, FGF, and GLP-1.

[0043] The composition comprising genetically modified islet organoids and silk matrix can be prepared in various ways.The genetically modified islet organoids can be prepared separately at first, and then placed on silk matrix and allowed to attach.Alternatively, the genetically modified islet organoids can be prepared separately at first, and then placed in silk solution, and allowed to form silk matrix containing genetically modified islet organoids.In a further preferred embodiment, the method for providing the composition comprising genetically modified islet organoids and silk matrix comprises:(i) providing an aqueous solution of silk protein that can be assembled into water-insoluble macrostructures;(ii) preparing an aqueous mixture of a sample of genetically modified islet cells with silk protein that optionally further comprises laminin;(iii) allowing silk protein to assemble into water-insoluble macrostructures in the presence of islet cells, thereby forming a 3D silk matrix for eukaryotic cells;and(iv) maintaining and differentiating genetically modified islet cells in silk matrix under suitable conditions for obtaining genetically modified islet organoids. In further embodiments, the method can include a step in which the genetically modified pancreatic islet organoids are detached from the silk matrix and allowed to grow in suspension, if desired.

[0044] In a third aspect, the present disclosure provides the use of the genetically modified islet organoid of the first aspect of the present disclosure or the composition of the second aspect of the present disclosure for the treatment of disorder.In a fourth aspect, the present disclosure provides a method for the treatment of disorder, comprising transplanting the genetically modified islet organoid or composition of any embodiment or combination of embodiments of the present disclosure to a subject with disorder in an amount effective for treating said disorder.As discussed above, the present disclosure provides the novel and surprising finding that islet organoid can be genetically modified to express any polypeptide, including non-pancreatic polypeptide, and that these genetically modified islet organoids can be used to treat diseases that are treatable using any polypeptide, including non-pancreatic polypeptide produced by genetically modified islet organoid.

[0045] As used herein, the term "subject" is any subject for which treatment is desired, such as a mammalian subject, including but not limited to humans, cows, dogs, cats, guinea pigs, rabbits, rats, mice, cattle, horses, etc. In one embodiment, the subject is a human. As defined herein, a "subject in need thereof" is a subject suffering from a disorder associated with reduced levels of expression, or absent expression, of a polypeptide that is not endogenously expressed by pancreatic islets.

[0046] In one embodiment of the use and method of the present disclosure, genetically modified pancreatic islet organoid or composition may be transplanted into subject.Transplantation can be achieved through any suitable procedure, and genetically modified pancreatic islet organoid can be transplanted into any suitable area of ​​subject, including but not limited to subject's eye, pituitary gland, pancreas, small intestine, stomach, brain, kidney, parathyroid gland, duodenum, thyroid gland, liver, heart, ovary, testicle, fat or skin.In one embodiment, genetically modified pancreatic islet organoid is transplanted into subject's eye.

[0047] The use and method can be used to treat any disorder associated with or caused by a reduced expression level or complete absence of circulating polypeptide.In various embodiments, the disorder to be treated and the polypeptide expressed by the genetically modified islet organoid of the present disclosure to treat the disorder are listed in the same row in Table 1 below.Thus, for example, if the genetically modified islet organoid expresses thyroid stimulating hormone, the use and method may include treating a subject with hypothyroidism (see the second row in Table 1).Similarly, if the genetically modified islet organoid expresses prolactin, the use and method may include treating a subject with hypoprolactinemia (see the third row in Table 1).Thus, by displaying each row in Table 1, it will be clear to those skilled in the art how to determine which genetically modified islet organoid expressing which polypeptide may treat which disorder.

[0048] [Table 1] TIFF2024516364000002.tif242160TIFF2024516364000003.tif242162TIFF20245163640 00004.tif242161TIFF2024516364000005.tif242161TIFF2024516364000006.tif117162

[0049] In certain embodiments, the disorders include hypoprolactinemia, hypocalcemia, Prader-Willi syndrome, obesity, hypertriglyceridemia, lipodystrophy (Beardinelli-Seip syndrome, Lawrence syndrome, and Barackel-Siemons syndrome), hemophilia, Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidoses, Wolman disease, hereditary angioedema, chronic neutropenia, renal anemia, thalassemia a beta, hemophilia-like disorders including hemophilia A and B, phosphatasia, pulmonary edema, and pulmonary edema. von Willebrand's disease, Factor XIII deficiency, Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic Uremic Syndrome (HUS), Phenylketonuria, Emphysema, Hypophosphatasia, Cystic Fibrosis, Depression, Pain, Neurotransmission Related Disorders, Parkinson's Disease, ADD, Anxiety, Memory Loss, Rheumatoid Arthritis (RA), Cryopin-Associated Periodic Syndrome (CAPS), and Alzheimer's Disease, Diabetes, Metabolic Syndrome, Schizophrenia, Autism, Seizure Related Disorders, Eating Disorders, Stroke Les disorders, gastrointestinal diseases, leukemia, cancer, cardiovascular diseases, Lyme disease, tropical diseases, brain cancer, necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcers and congenital microvillous atrophy, spondylolisthesis, posterolateral arthrodesis, osteoporosis, spinal fusion, fracture healing, immune disorders, autoimmune disorders, cardiovascular diseases, blood clotting disorders, Alport syndrome, epidermolysis bullosa, type 1 and type 2 diabetes, severe insulin resistance syndrome, Crohn's disease, arthritis, juvenile chronic arthritis, cystic fibrosis, neovascularization of the eye, Include duct formation, growth hormone deficiency, wound healing, tissue repair, neutropenia, neurofibroma, diabetic neuropathy, neurodegenerative and neuromuscular disease, Huntington's disease, chronic inflammatory disease, ischemic and hemorrhagic stroke, traumatic brain injury (TBI), dementia, multiple sclerosis, motor neuron disease, amyotrophic lateral sclerosis, ataxia, muscular dystrophy, spinal muscular atrophy, stroke, spinal cord injury, Charcot-Marie-Tooth disease and progressive supranuclear palsy.In other specific embodiments, the polypeptide expressed by the genetically modified pancreatic islet organoid of the present disclosure for treating these disorders and disorders are listed in the same row in Table 1.

[0050] In one particular embodiment, organoids may comprise pancreatic beta cells comprising recombinant expression vectors, and the expression vector comprises nucleic acid encoding leptin under the control of insulin promoter. Organoids may be surgically transplanted, for example, into the anterior chamber of the eye or into the brain. Transplantation of organoids results in the production and release of leptin from pancreatic beta cells that do not normally express leptin. This embodiment can result in lowering blood glucose and insulin levels, which can be used to treat obesity, hypertriglyceridemia, lipodystrophy (Belardinelli-Seip syndrome, Lawrence syndrome, and Baraker-Siemons syndrome).

[0051] In other non-limiting embodiments, one or more of GLP-1, GLP-1 analogs that are resistant to degradation by DPP4, and ligands that can interact with the GLP-1 receptor and other receptors (including the GIP receptor and the glucagon receptor) can be expressed and released from the genetically modified pancreatic islet organoids after transplantation, for example into the eye or brain. Examples of ligands capable of interacting with GLP-1, GLP-1 analogs, GLP-1 receptors and other receptors include, but are not limited to, dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, LY2944876 / TT401, SAR425899, MEDI0382, HM12525A / JNJ-64565111, ZP2929 / BI 456906, MK-8521, NN9277, RG7697 / NNC0090-2746, DA2GIP-Oxm, [dA2]GLP-1 / glucagon, Y1-dA2-I12-N17-V18-I27-G28, 29-glucagon, MAR423, and HM15211. Such embodiments can be used to treat any condition associated with GLP-1, GIP, glucagon, and / or oxymodulin, including but not limited to diabetes, obesity, metabolic syndrome, and neurological disorders, including but not limited to Parkinson's disease.

[0052] In other non-limiting examples, the pancreatic organoid can comprise beta cells comprising recombinant expression vectors, the expression vectors comprising nucleic acids encoding one or more of GLP-1, GLP-1 analogs, and / or ligands that can interact with GLP-1 receptor. In various examples of this embodiment, the recombinant pancreatic organoids comprising beta cells expressing GLP-1 or GLP-1 analogs can be surgically transplanted into any location, including the anterior chamber of the eye or the brain. Surgical transplantation into the brain can include transplantation into the dura mater, either outside or inside the dura mater. In various examples of this embodiment, the recombinant expression vector can comprise any suitable promoter, including but not limited to insulin and glucagon promoters. In this embodiment, the beta cells are genetically modified to secrete GLP-1, and thus can function as a positive autocrine feedback signal that will increase insulin secretion from the beta cells when blood glucose concentration is high. In one particular embodiment, genetically modified pancreatic organoid comprises pancreatic beta cell comprising recombinant expression vector, and expression vector comprises nucleic acid encoding one or more of GLP-1, GLP-1 analogue, and / or ligand that can interact with GLP-1 receptor under the control of insulin promoter.In this particular embodiment, genetically modified pancreatic organoid can be transplanted into brain and used to treat diabetes, obesity, metabolic syndrome and neurological disorders, including Parkinson's disease.

[0053] Any suitable expression of the polypeptide in a subject can confer a benefit in treating the disorder (i.e.: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as a percentage of the expression level in a subject not having the disorder).

[0054] Example 1: Genetically modified pancreatic islet organoids in the eye as a production site for blood-derived peptides / proteins method animal. Both female B6.Cg-Lep mice were on a C57BL / 6J background. ob / J(ob / ob) mice and B6.BKS(D)-Lepr db A / J mice were purchased at 2 months of age from Charles River Laboratories (Wilmington, MA, USA). After delivery, the mice were acclimated to the animal facility for 1 week before the start of the experiments. All animals were group-housed on a 12 / 12 h light / dark cycle with free access to food (chow feed R70 from Lantmannen, Stockholm, Sweden) and water. All experiments were performed in accordance with the Karolinska Institutet guidelines for the care and use of animals in research and were approved by the Animal Ethics Committee.

[0055] Leptin treatment of ob / ob mice: ob / ob mice were treated daily with a single intraperitoneal injection of recombinant human leptin protein (1.5 μg / g body weight, R&D Systems, Minneapolis, USA) for 5 weeks from the day of arrival.

[0056] Expression vector construction:Mouse leptin cDNA was obtained from pCMV6.mouse leptin (OriGene, #MC208876) and subcloned into pTRE-tight (Clonetech) to generate pTRE-tight.m-leptin. An IRES-mCherry™ sequence was then inserted downstream of the m-leptin cDNA to obtain pTRE-tight.m-leptin-IRES-mCherry™. To generate pENTR.TRE-tight.m-leptin-IRES-mCherry™ / RIP1.DsRed2™, the rbGK.EGFP cassette was replaced in pENTR.rbGK.EGFP / RIP1.DsRed2™ (Paschen et al., FASEB J 2019) by TRE-tight.m-leptin-IRES-mCherry™. The DsRed™ cDNA was then exchanged in pENTR.TRE-tight.mLeptin-IRES-mCherry™ / RIP1.DsRed2™ by the TetOFF-IRES-ZsGreen™ cassette obtained from pTetOFF-Dual(green) (Clontech), thus generating pENTR.TRE-tight.mLeptin-IRES-mCherry™ / RIP1.TetOFF-IRES-ZsGreen™. All constructs were verified by DNA sequence analysis. The TRE-tight.mLeptin-IRES-mCherry™ / RIP1.TetOFF-IRES-ZsGreen™ cassette was transferred by Gateway technology into the promoterless adenoviral plasmid pAd / PL-DEST (Thermo Fisher Scientific). The ViraPower™ Adenovirus Expression System (Thermo Fisher Scientific) was used to generate a replicon-deficient adenovirus, designated vAd / RIP-mLeptin-OFF, which was used to transduce cells.

[0057] Isolation of pancreatic islets: Pancreatic islets were transfected with B6.BKS(D)-Lepr dbPancreatic islets were isolated from db / J mice (db / db). Pancreatic islets were prepared from mice by pancreatic duct injection of collagenase (F. Hoffmann-La Roche, Basel, Switzerland) and, after digestion, handpicked under a stereomicroscope MZ6 (Leica Microsystems, Wetzlar, Germany). The islets were then cultured at 5% CO2 and 37°C in RPMI-1640 medium (RPMI medium) with a final concentration of 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U / ml penicillin, and 100 mg / ml streptomycin (RPMI medium).

[0058] Pancreatic Islet Organoid Generation: Islets were collected in 1.5 ml Eppendorf tubes (250 islets / tube), digested with Accutase™ (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 37°C, and centrifuged at 500 rpm. 4 × 10 leptin-encoding adenovirus per ml were incubated in RPMI medium in suspension culture dishes for 1 h at 5% CO2 and 37°C. 6 Pancreatic islet cells were transduced with plaque forming units and then washed twice with excess RPMI medium to remove excess adenovirus.

[0059] 2500 islet cells were seeded into each well of a Nunclon Sphera™ 96U bottom plate (Thermo Scientific, Leicestershire, UK). Islet organoids were allowed to form for 5 days, after which the organoids were transferred to suspension culture dishes.

[0060] In Vitro Imaging of Intraocular Islet Organoids: Pancreatic islet organoids were imaged as three-dimensional stacks with a step size of 3 μm using an inverted confocal laser scanning microscope (TCS SP8; Leica Microsystems). ZsGreen™ fluorescent protein was excited at 488 nm and fluorescence was detected at 505–535 nm, while mCherry™ was excited at 561 nm and fluorescence was detected at 580–650 nm. Backscatter signal (reflection) from the 561 nm excitation was collected at 555–565 nm.

[0061] Leptin secretion from pancreatic islet organoids in vitro:Pancreatic islet organoids were cultured in vitro in suspension culture dishes in RPMI medium. For glucose-dependent leptin secretion measurements, organoids were placed in 4-well plate suspension dishes containing RPMI medium with 3 mM glucose for 1.5 h. Then, organoids were placed in new wells containing medium with 3 mM glucose for 30 min, followed by 16 mM glucose for 30 min. Then, organoids were returned to suspension culture dishes containing regular RPMI medium and continued to be cultured. The medium from the wells of the 4-well plate was collected in an Eppendorf tube and centrifuged for 1 min to pellet viable organoid fragments. The supernatant was collected in a new tube and kept at -20°C until analysis.

[0062] Transplantation of islet organoids into ACE: Using a technique previously described by Speier et al. (Speier et al., Nat Protoc 2008), islet organoids were transplanted into the ACE of three (first experimental set) and seven (second experimental set) ob / ob recipients. Briefly, under anesthesia, a small hole was made in the cornea with a 27-gauge needle, after which the organoids were transplanted into the ACE using a glass cannula. Great care was taken to avoid bleeding and damage to the iris. For postoperative analgesia, mice were injected subcutaneously with Temgesic™ (0.1 ml / kg; RB Pharmaceuticals, Berkshire, UK). Each mouse in the transplant group received 100 organoids / eye. Three ob / ob mice (first experimental set) and five ob / ob mice (second experimental set) were used as controls and were not transplanted.

[0063] In vivo imaging of intraocular islet organoid grafts:Islet organoid grafts were imaged in vivo starting from 9 weeks after transplantation. An upright confocal laser scanning microscope (TCS SP5; Leica Microsystems) equipped with a long working distance immersion objective (HXC-APO103 / 0.30 numerical aperture; Leica Microsystems) and a custom-made stereotaxic head holder, allowing the mouse eye containing the transplanted islets to be positioned towards the objective, was used. Viscotear™s (Thea Nordic, Örebro, Sweden) was used as immersion fluid between the eye and the objective, and isoflurane was used to anesthetize the mice during in vivo imaging. Grafts were imaged as three-dimensional stacks with a step size of 3 μm. ZsGreen™ fluorescent protein was excited at 488 nm and fluorescence was detected at 505–535 nm. mCherry™ was excited at 561 nm and fluorescence was detected at 580–650 nm. Backscatter signal (reflection) from 561 nm excitation was collected at 555–565 nm. After imaging, mice were allowed to recover from anesthesia. Additionally, beginning 6 weeks after transplantation, overview images of the grafts were obtained while the mice were under anesthesia using a digital camera connected to a Leica M60 stereomicroscope.

[0064] Doxycycline treatment of animals: To stop leptin production from the islet organoid grafts, three animals in the transplant group were treated with doxycycline: sterile doxycycline hydrochloride in PBS was administered intraperitoneally (ip) five times over a 10-day period (50 μg / kg / mouse).

[0065] Weight and fasting and non-fasting blood glucose: After 6 hours of food refusal, body weight and fasting blood glucose were measured. Non-fasting blood glucose was measured at 4 pm with full access to food.

[0066] Intraperitoneal glucose tolerance test (ipGTT):To determine glucose tolerance, blood glucose levels were measured in mice fasted for 6 h at basal state (0 min) and 10, 30, 60, and 120 min after glucose injection (2 g / kg body weight ip, dissolved in PBS). Results were expressed as area under the curve (AUC) of the ipGTT. Glucose concentrations were measured using the Accu-Chek™ Aviva Monitoring System (F. Hoffmann-La Roche, Basel, Switzerland).

[0067] Plasma and aqueous fluid samples: Blood samples were taken from nonfasted animals, collected in Microvette CB300 EDTA / PK100 tubes (Sarstedt, Nümbrecht, Germany), centrifuged to obtain plasma, and stored at −20° C. until use. Aqueous humor samples were obtained at the end of the experiment and kept at −20° C. until use.

[0068] Insulin and C-peptide measurements: Plasma insulin and C-peptide levels were analyzed using ultrasensitive mouse ELISA kits (Crystal Chem, Elk Grove Village, IL, USA).

[0069] Leptin Measurement: Leptin was measured in cell culture medium, plasma and aqueous fluid samples using a Mouse / Rat Leptin Quantikine™ ELISA kit (R&D Systems, Minneapolis, USA).

[0070] Tissue extraction and sectioning:Eyes were obtained and sectioned to confirm and complement the data obtained in vivo. Mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Eyes were extracted and fixed in 4% paraformaldehyde for 1 week. Before cryopreservation, eyes were processed in a sucrose density gradient [10–30% (wt / vol) sucrose in PBS containing 0.01% (wt / vol) sodium azide and 0.02% (wt / vol) bacitracin], embedded in OCT-compound (Tissue-Tec, Sakura Finetech, Torrance, CA, USA), frozen on dry ice, and stored at −80°C until use. Then, 20 μm-thick cryosections of the anterior part of the eye were collected on SuperFrost™ Plus microscope slides (VWR International, Radnor, PA, USA) and kept at −20°C until use.

[0071] Immunofluorescence in eye sections: For immunostaining, eye sections were equilibrated to room temperature, washed, blocked, and then incubated with primary antibodies, goat anti-m-leptin (R&D Systems, Minneapolis, USA) and rabbit anti-C-peptide (Cell Signaling, Danvers, MA, USA), in the presence of 0.1% Triton X-100 and 10% serum. After washing, secondary antibodies, anti-goat Alexa 633 and anti-rabbit Alexa 594, respectively (Thermo Fisher Scientific), were applied, and after repeated washing, fixation was performed with medium containing DAPI (Thermo Fisher Scientific) for nuclear counterstaining. Imaging was performed using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems) with the following excitation settings: DAPI-excitation 405 nm, detection 450-470 nm; ZsGreen™ excitation 488 nm, detection 500-525 nm; mCherry™ 548 nm, detection 560-580 nm; Alexa594 (C-peptide stain) excitation 594 nm, detection 600-620 nm; Alexa633 (leptin stain) excitation 633 nm, detection 640-680 nm. Imaging was performed using frame-to-frame sequential imaging to avoid spectral overlap.

[0072] statistics:Values ​​are expressed as mean ± SEM. Two-tailed unpaired t-test was used to determine statistical significance between different treatment groups. Statistical significance was used as follows: *P<0.05, **P<0.01, ***P<0.001. Origin 2015 64-bit (OriginLab, Northampton, MA, USA) and Excel (Microsoft, Redmond, WA, USA) were used for statistical analysis.

[0073] result Generation of a Tet-off based beta cell specific adenoviral expression construct for mouse leptin and its in vitro evaluation: An adenoviral vector, vAd-RIP-leptin-OFF (Figure 1A), was generated that contains two expression cassettes located in opposite orientations and separated by a "transcription block" sequence to allow independent expression. The first expression cassette allows rat insulin-1 promoter-driven expression of the synthetic transcription factor rTA (Tet-off) and the green fluorescent protein ZsGreen™ in pancreatic beta cells. The second expression cassette consists of a TRE-tight promoter-driven mouse leptin-IRES-mCherry™ cassette. Binding of rTA to the TRE-tight promoter induces expression of leptin and the red fluorescent protein mCherry™ in beta cells in the absence of doxycycline, whereas addition of doxycycline stops expression of the two proteins. The IRES elements in the two expression cassettes allow simultaneous expression of rTA with ZsGreen™ and leptin with mCherry™. Thus, "green" and "red" serve as visual readouts for the expression of rTA and leptin, respectively, in beta cells.

[0074] For in vitro evaluation, B6.BKS(D)-Lepr dbTransduced organoids made from islets of 1 / J mice (from the same batch used for transplantation) were used. Detection of green and red fluorescence by laser scanning confocal microscopy of islet organoids showed that both expression cassettes were activated in beta cells (Figure 1B and Figure 1C). Organoids secreted leptin in culture medium, and secretion was higher at higher glucose concentrations: 7.02 + / - 1.76 pg / organoid / h at 3 mM glucose vs. 12.94 + / - 2.83 pg / organoid / h at 16 mM glucose (stimulation index: 1.93 + / - 0.14).

[0075] Ectopic leptin production by islet organoid grafts in ACE improves metabolic phenotype in ob / ob mice. To test whether intraocular leptin production by islet organoids affects metabolic phenotype in ob / ob mice, the following was considered. Since leptin deficiency after weaning leads to rapid development of ob / ob phenotype, including robust beta cell proliferation, in both original in situ islets and islets transplanted in ACE, and leptin treatment slows down this process (Ilegems et al., PNAS 2013), ob / ob mice were treated with daily intraperitoneal injections of leptin (1.5 μg / g body weight / day) immediately after arrival from the vendor and until 4 weeks after transplantation of islet organoid grafts, i.e. after their complete engraftment. For organoid generation, islets from leptin receptor-deficient mice, i.e. ob / ob recipient mice (B6.Cg-Lep ob db / db mice (B6.BKS(D)-Lepr / J) with a B6 genetic background to match the B6 background of the db By using islets from db / db mice, the potential negative feedback of beta cell-produced leptin was abrogated. Therefore, we isolated islets from db / db mice, disaggregated them, transduced the islet cells with vAd-RIP-leptin-OFF, and formed islet organoids by self-assembly.

[0076] In the first set of experiments, 130 leptin-expressing db / db islet organoids treated with leptin from day 15 pre-transplant until day 28 post-transplant were transplanted into the ACE of female ob / ob mice (n=3, see Fig. 2A, Fig. 2B), and leptin-treated female ob / ob mice (n=3) in which no islet organoids were transplanted served as a control group. Early studies using non-transformed islets as a control for transplantation showed overgrowth of these islet grafts (Fig. 2C), which required termination of the experiment for ethical reasons, so non-transformed islet organoids were not used in the control group. Examination of islet organoids maintained in vitro (Fig. 1B), as well as organoids transplanted into the ACE in vivo (Fig. 2B), showed expression of both green and red fluorescent proteins, suggesting that both the rTA-IRES-ZsGreen™ and leptin-IRES-mCherry™ expression cassettes were expressed in beta cells. No difference in the body weight of the mice between both groups (Figure 2D) was observed. However, changes in the glucose tolerance test (ipGTT) were observed, which started 10 days after the cessation of leptin treatment and became prominent 25 days after the cessation of leptin treatment (Figure 2E). Furthermore, a trend towards a decrease in fasting blood glucose was observed in the group transplanted with leptin-producing organoids, which was prominent 35 days after the cessation of leptin treatment (Figure 2F), as well as a trend towards a decrease in plasma insulin (Figure 2G) and insulin C-peptide (Figure 2H) levels, both measured at the end of the experiment. Finally, leptin (11.375 + / - 3.211 ng / ml) was detected in the aqueous humor at the end of the experiment in the transplanted group, whereas leptin was undetectable in the control group. Leptin was undetectable in the plasma from both groups.

[0077] In a second set of experiments, 200 leptin-expressing db / db islet organoids treated with leptin from day 7 before transplantation until day 28 after transplantation were transplanted into the ACE of female ob / ob mice (n=7, see Fig. 3A, Fig. 3B), and leptin-treated female ob / ob mice (n=5) in which no islet organoids were transplanted served as a control group. Examination of islet organoids maintained in vitro (Fig. 1C), as well as organoids transplanted into ACE in vivo (Fig. 3B), showed expression of both green and red fluorescent proteins, suggesting that both rTA-IRES-ZsGreen™ and leptin-IRES-mCherry™ expression cassettes were expressed in beta cells. Importantly, in this experiment, mice transplanted with leptin-producing organoids showed significant differences in body weight from day 15 after cessation of leptin treatment (Fig. 3C). Significant changes in ipGTT were observed starting from day 19 after the cessation of leptin treatment (Figure 3D). In addition, a decrease in fasting blood glucose in the group transplanted with leptin-producing organoids (Figure 3E, noticeable at days 19 and 68 after the cessation of leptin treatment), a significant decrease in plasma insulin (from day 14 after the cessation of leptin, Figure 3F), and a significant decrease in plasma C-peptide levels (from day 8 after the cessation of leptin, Figure 3G) were observed. Finally, leptin (6.05 + / - 2.91 ng / ml) was detected in the aqueous humor at the end of the experiment in that group, whereas leptin was undetectable in the control group. The value of leptin in the plasma of the transplanted group was 185.62 ± 86.11 pg / ml from day 14 after the cessation of leptin treatment until the end of the experiment. Leptin was undetectable in the plasma of the control group.

[0078] Doxycycline treatment abolishes ectopic leptin expression.Three animals in the transplant group were treated with doxycycline (five intraperitoneal injections of 50 μg dox / kg / mouse over 10 days) to stop leptin production from transplanted organoids. Before doxycycline treatment in the grafts, both ZsGreen™ and mCherry™ expression were monitored. Following doxycycline treatment, mCherry™ expression was not detectable in the grafts of these animals (Figure 4Bc), and leptin expression was not detectable by immunohistochemistry (Figure 4Dc). In the grafts of non-doxycycline treated animals, mCherry™ and leptin expression were detectable (Figure 4A and Figure 4C). More importantly, leptin was not measurable in the aqueous humor of doxycycline treated animals (n=3).

[0079] Experimental data provide evidence that genetically modified pancreatic islet organoids can function as production sites for proteins / peptides.

[0080] Experiment 2: GLP-1 and GLP-1 analogs, leptin GLP-1, GLP-1 analogs that are resistant to degradation by DPP4, and ligands that can interact with other receptors, including the GLP-1 receptor, the GIP receptor, and the glucagon receptor, as well as incretins, can be expressed and released from the genetically modified pancreatic islet organoids.

[0081] We investigate the effects of genetically modified islet organoids transplanted (both metabolic and reporter transplants) into the anterior chamber, brain, and kidney capsule. These transplanted islet organoids secrete either GLP-1 or leptin.

[0082] GLP-1 is typically not expressed in significant amounts by pancreatic islets, but is expressed by L-cells in the gastrointestinal tract. Injected or orally administered GLP-1, or synthetic ligands for the GLP-1 receptor, can have an effect on glucose control in the body through interactions with GLP-1 receptors in beta cells, as well as central and gastric GLP-1 receptors.

[0083] We have shown that direct release of GLP-1, GLP-1 analogs and other ligands capable of interacting with the GLP-1 receptor, within the pancreatic islets via a local autocrine feedback loop, can activate insulin release.

[0084] Experiments include adenovirus production, pseudoislet generation and viral transduction.

[0085] method Adenovirus production. Adenovirus or adeno-associated virus vectors are prepared using the Gateway™ cloning system (Thermo Fisher Scientific). (7-37) The gene sequence is placed under the CMV promoter or under the rat insulin promoter RIP1 sequence, resulting in non-cell specific expression or ensuring targeted expression in insulin producing cells, respectively.

[0086] Viral particle quantification is performed by real-time PCR on a QuantStudio 5™ system (Thermo Fisher Scientific) using SYBR Green (Thermo Fisher Scientific) and primers specific for the adenovirus coding sequence.

[0087] Pseudoislet generation and viral transduction. For the generation of pseudoislets, islets are dissociated to single cells by enzymatic digestion using Accutase™ (Sigma-Aldrich) for 10 min at 37°C. Cells are counted using an automated cell counter (BRIPio-Rad) and resuspended at a density of 12500 cells / ml in serum-supplemented culture medium. The cell suspension is distributed into ultra-low attachment 96-well plates (PerkinElmer) using 200 μl of cell suspension per well. 1 × 10 cells per ml of cell suspension are added before distributing into plates. 6 Transduction is performed by adding 1000 x 1000 viral particles. Pseudoislets are formed according to the manufacturer's instructions and harvested after 7 days.

[0088] Planned studies. In addition to glucose homeostasis, the reporter islets are evaluated for their function in demonstrating sustained function. Blood glucose, hemoglobin A1c, and insulin release and levels are measured. Blood glucose levels are shown to be lower in subjects receiving transplants of GLP-1-secreting islet organoids than in subjects that did not. Insulin release is improved in treated animals. Transplanted GLP-1-secreting islet organoids are used in the treatment of diabetes and obesity.

[0089] Genetically modified islet organoids expressing several different incretins simultaneously, including GLP-1, somatostatin and GIP, are prepared by mixing in defined ratios and then allowing the reassortment of different phenotypes of islet cells transfected with different target loads, showing preferential release after feeding (target load in genetically modified beta cell phenotype), during fasting (target load in alpha cell phenotype) and basal release, depending on the desired time-profile associated with the target load.

[0090] Genetically modified pancreatic islet organoids were transplanted into the brain to show localized release of polypeptides including GLP-1 and leptin. The grafts were placed either just outside or / and within the dura mater of the brain.

[0091] result The transplanted pancreatic islet organoid secreting GLP-1 is used to treat diabetes, obesity, metabolic syndrome and neurodegenerative diseases such as Parkinson's disease.It is shown that the transplanted pancreatic islet organoid becomes vascularized and innervated after transplantation, and secretes GLP-1.

[0092] Brain-transplanted islet organoids secreting leptin are used to treat obesity, hypertriglyceridemia, and lipodystrophy (Beardinelli-Seip syndrome, Lawrence syndrome, and Baraker-Siemons syndrome).

[0093] For the leptin-secreting islet organoid transplanted into brain, carry out the same measurement as in Example 1. It is shown that the transplanted islet organoid becomes vascularized and innervated, and secretes leptin.Furthermore, this secretion improves the metabolic phenotype in ob / ob mice, including reducing blood glucose and insulin levels.

[0094] References [Table 2]

Claims

1. A genetically modified pancreatic islet organoid comprising a recombinant expression vector, the recombinant expression vector comprising (a) a nucleic acid sequence encoding a polypeptide, and (b) a suitable control sequence operably linked to the nucleic acid sequence.

2. The genetically modified pancreatic islet organoid of claim 1, wherein the recombinant expression vector is present in a pancreatic islet organoid cell that does not endogenously express the polypeptide.

3. The genetically modified pancreatic islet organoid of claim 1 or 2, wherein the polypeptide is not endogenously expressed in pancreatic islet cells.

4. The genetically modified pancreatic islet organoid of claim 1, wherein the polypeptide is a therapeutic polypeptide or an analog thereof.

5. The genetically modified pancreatic islet organoid of claim 4, wherein the therapeutic polypeptide is selected from the group consisting of polypeptide hormones and enzyme replacement proteins.

6. The genetically modified pancreatic islet organoid of claim 5, wherein said therapeutic polypeptide comprises a polypeptide hormone selected from the group consisting of thyroid stimulating hormone (TSH), prolactin, parathyroid hormone (PTH), leptin, follicle stimulating hormone (FSH) and growth hormone (GH) or their analogs.

7. The genetically modified pancreatic islet organoid of claim 5, wherein said therapeutic polypeptide comprises a polypeptide hormone selected from the group consisting of thyrotropin releasing hormone (TRH), renin, gastrin, vasoactive intestinal peptide (VIP), vasopressin (ADH), oxytocin (OXY), melanocyte stimulating hormone (MSH), calcitonin, cholecystokinin (CCK), atrial natriuretic peptide (ANP), angiotensin, amylin, and adrenocorticotropic hormone (ACTH), or their analogs.

8. The therapeutic polypeptide comprises an enzyme replacement protein, the enzyme replacement protein being selected from the group consisting of factor VII (eptacog alfa), factor VIII, factor IX, factor XIII (catridecacog alfa), von Willebrand factor, taliglucerase alfa, agalsidase alfa or beta, imiglucerase, velaglucerase alfa, alglucosidase alfa, galsulfase, dornase alfa, laronidase, conestat alfa (C1 esterase inhibitor), pegloticase alfa-1-proteinase inhibitor, asfotase alfa (Strensic), idursulfase, elosulfase alfa, valiase, sebelipase alfa, epoetin theta, 6. The genetically modified pancreatic islet organoid of claim 5, wherein the selected from the group consisting of teta) (Eporatio), beta (NeoRecormon), zeta (Retacrit), darbepoetin alfa (Aranesp), luspatercept (Reblozyl), filgrastim, lenograstim, and von Willebrand factor-cleaving protease, or analogs thereof.

9. The therapeutic polypeptide may be an enkephalin, endorphin, substance P, neurotensin, neuropeptide-Y, bombesin, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cholinergic differentiation factor, cardiotrophin-1, oncostatin M, tumor necrosis factor (TNF), Neu differentiation factor, heregulin, acetylcholine receptor-inducing activity, glial cell growth factor (GGF), glial cell line-derived neurotrophic factor (GDNF), artemin, neurturin, persephin, bone morphogenetic protein-1 (OP-1), bone morphogenetic protein (BMP), growth differentiation factor, ephrin, epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDG F), hepatocyte growth factor (HGF), granulocyte colony stimulating factor (G-CSF), serine protease inhibitor, protease nexin-1, hedgehog family of inducible proteins, agrin, laminin 2, neuroimmunophilin, pigment epithelium-derived factor (PEDF), activity-dependent neuroprotective protein (ADNP), neuritin (activity-induced neurotrophic factor), angiogenic growth factor, brain dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), peptide-6, davunetide (derived from ADNP), and cerebrolysin.

10. The genetically modified pancreatic islet organoid of claim 1, wherein the recombinant expression vector comprises a viral vector.

11. The genetically modified pancreatic islet organoid of claim 10, wherein the viral vector is selected from the group consisting of retroviral vector, lentiviral vector, murine leukemia virus (MMLV) vector, murine stem cell virus (MSCV) vector, adenoviral vector, herpes simplex virus vector, baculovirus vector and adeno-associated virus vector.

12. The genetically modified pancreatic islet organoid of claim 1, wherein the expression vector comprises a non-viral vector.

13. The genetically modified pancreatic islet organoid of claim 12, wherein the non-viral vector comprises gene editing.

14. The genetically modified pancreatic islet organoid of claim 13, wherein the gene editing comprises a CRISPR / Cas9 system.

15. The genetically modified pancreatic islet organoid of claim 1, wherein the suitable control sequence is pancreatic tissue specific and selected from the group consisting of midkine (MK) promoter, cyclooxygenase-2 (Cox2)M promoter, Cox 2L promoter, vascular endothelial growth factor (VEGF) promoter, caveolin 1 promoter, fms-like receptor tyrosine kinase 1 (FLT-1) promoter, sloopy paired 1 (SLP-1) promoter, gastrin releasing peptide (GRP) promoter, epithelial glycoprotein 2 (EGP-2) promoter, insulin promoter, and glucagon promoter.

16. 16. The genetically modified pancreatic islet organoid of claim 15, wherein the recombinant expression vector comprises an adeno-associated virus vector, and the insulin promoter is RIP1 or RIP2.

17. The genetically modified pancreatic islet organoid of claim 1, wherein the suitable control sequence is selected from the group consisting of cytomegalovirus (CMV) promoter, chicken beta-actin (CAG) promoter, Rous sarcoma virus (RSV), Simian virus 40 (SV40), mammalian elongation factor 1 alpha (EF1 alpha) promoter, muscle creatine kinase (MCK) promoter, human phosphoglycerate kinase 1 (PGK1) promoter, and tetracycline response element (TRE)-tight promoter.

18. A composition comprising the genetically modified pancreatic islet organoid of claim 1 and a silk matrix.

19. 20. The composition of claim 18, wherein the silk is spider silk.

20. 20. The composition of claim 18 or 19, wherein the silk is functionalized with a cell-binding motif.

21. Use of the genetically modified pancreatic islet organoid of claim 1 or the composition of claim 18 for treating a disorder.

22. The disorder may be hypoprolactinemia, hypocalcemia, Prader-Willi syndrome, obesity, hypertriglyceridemia, lipodystrophy (Beardinelli-Seip syndrome, Lawrence syndrome, and Barackel-Siemons syndrome), hemophilia, Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidoses, Wolman disease, hereditary angioedema, chronic neutropenia, renal anemia, thalassemia a beta, hemophilia-like disorders including hemophilia A and B, von Willebrand disease, factor X syndrome, Factor III deficiency, thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), phenylketonuria, emphysema, hypophosphatasia, cystic fibrosis, depression, pain, neurotransmission-related disorders, Parkinson's disease, ADD, anxiety, memory loss, rheumatoid arthritis (RA), cryopin-associated periodic syndrome (CAPS), and Alzheimer's disease, diabetes, metabolic syndrome, schizophrenia, autism, seizure-related disorders, eating disorders, stress disorders, gastrointestinal disorders, leukemia, cancer , cardiovascular disease, Lyme disease, tropical diseases, brain cancer, necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcers and congenital microvillous atrophy, spondylolisthesis, posterolateral arthrodesis, osteoporosis, spinal fusion, fracture healing, immune disorders, autoimmune disorders, cardiovascular disease, blood clotting disorders, Alport syndrome, epidermolysis bullosa, type 1 and type 2 diabetes, severe insulin resistance syndrome, Crohn's disease, arthritis, juvenile chronic arthritis, cystic fibrosis, ocular neovascularization, growth hormone deficiency, wound healing 22. The use of claim 21, wherein the therapeutic agent is selected from the group consisting of: inflammatory bowel disease, chronic inflammatory diseases, chronic myelopathy, chronic inflammatory bowel disease, chronic myelopathy ...

23. A method for treating a disorder, comprising transplanting into a subject having the disorder a genetically modified pancreatic islet organoid of claim 1 or a composition of claim 18 in an amount effective to treat the disorder.

24. The method of claim 23, wherein the genetically modified pancreatic islet organoid or composition is transplanted into the eye, pituitary gland, pancreas, small intestine, stomach, brain, kidney, parathyroid gland, duodenum, thyroid gland, liver, heart, ovary, testicle, fat, and / or skin of a subject in need thereof.

25. The disorder may be hypoprolactinemia, hypocalcemia, Prader-Willi syndrome, obesity, hypertriglyceridemia, lipodystrophy (Beardinelli-Seip syndrome, Lawrence syndrome, and Barackel-Siemons syndrome), hemophilia, Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidoses, Wolman disease, hereditary angioedema, chronic neutropenia, renal anemia, thalassemia a beta, hemophilia-like disorders including hemophilia A and B, von Willebrand disease, factor X syndrome, Factor III deficiency, thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), phenylketonuria, emphysema, hypophosphatasia, cystic fibrosis, depression, pain, neurotransmission-related disorders, Parkinson's disease, ADD, anxiety, memory loss, rheumatoid arthritis (RA), cryopin-associated periodic syndrome (CAPS), and Alzheimer's disease, diabetes, metabolic syndrome, schizophrenia, autism, seizure-related disorders, eating disorders, stress disorders, gastrointestinal disorders, leukemia, cancer , cardiovascular disease, Lyme disease, tropical diseases, brain cancer, necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcers and congenital microvillous atrophy, spondylolisthesis, posterolateral arthrodesis, osteoporosis, spinal fusion, fracture healing, immune disorders, autoimmune disorders, cardiovascular disease, blood clotting disorders, Alport syndrome, epidermolysis bullosa, type 1 and type 2 diabetes, severe insulin resistance syndrome, Crohn's disease, arthritis, juvenile chronic arthritis, cystic fibrosis, ocular neovascularization, growth hormone deficiency, wound healing 24. The method of claim 23, wherein the therapeutic target is selected from the group consisting of: healing, tissue repair, neutropenia, neurofibromas, diabetic neuropathy, neurodegenerative and neuromuscular diseases, Huntington's disease, chronic inflammatory diseases, ischemic and hemorrhagic stroke, traumatic brain injury (TBI), dementia, multiple sclerosis, motor neuron diseases, amyotrophic lateral sclerosis, ataxia, muscular dystrophies, spinal muscular atrophies, stroke, spinal cord injury, Charcot-Marie-Tooth disease, and progressive supranuclear palsy.

26. 22. The use of claim 21, wherein said nucleic acid sequence encodes a polypeptide listed in the left hand column of Table 1, and said subject has a disorder listed in the right hand column of Table 1 in the same row as said polypeptide.

27. 27. The use of claim 26, wherein the polypeptide comprises thyroid stimulating hormone (TSH) and the disorder comprises hypothyroidism.

28. 27. The use of claim 26, wherein the polypeptide comprises leptin and the disorder comprises obesity, hypertriglyceridemia, lipodystrophy, Belardinelli-Seip syndrome, Lawrence syndrome, and Barackel-Siemons syndrome.

29. 27. The use of claim 26, wherein the polypeptide comprises growth hormone and the disorder comprises growth hormone deficiency, Turner's syndrome, chronic renal failure, Prader-Willi syndrome, intrauterine growth restriction, or idiopathic short stature.

30. 27. The use of claim 26, wherein the polypeptide comprises alglucerase, imiglucerase, velaglucerase alfa, and / or taliglucerase alfa, and the disorder comprises Gaucher disease.

31. 27. The use of claim 26, wherein the polypeptide comprises agalsidase alpha and / or beta and the disorder comprises Fabry disease.

32. 27. The use of claim 26, wherein the polypeptide comprises alglucosidase alfa and the disorder comprises Pompe disease.

33. 27. The use of claim 26, wherein the polypeptide comprises laronidase, idursulfase, elosulfase alfa, and / or galsulfase, and the disorder comprises mucopolysaccharidosis.

34. 27. The use of claim 26, wherein the polypeptide comprises conestat alpha and the disorder comprises hereditary angioedema.

35. 27. The use of claim 26, wherein the polypeptide comprises factor VIII and / or factor IX and the disorder comprises a hemophilia-like disease.

36. 27. The use of claim 26, wherein the polypeptide comprises an alpha-1 proteinase inhibitor and the disorder comprises emphysema.

37. 27. The use of claim 26, wherein the polypeptide comprises dornase alfa and the disorder comprises cystic fibrosis.

38. 27. The use of claim 26, wherein the polypeptide comprises a soluble leptin receptor (sLR) and the disorder comprises obesity, hypertriglyceridemia, diabetes, or metabolic syndrome.

39. 27. The use of claim 26, wherein the polypeptide comprises an IL-1 receptor antagonist or anakinra and the disorder comprises rheumatoid arthritis (RA), an autoinflammatory disorder, systemic juvenile idiopathic arthritis, gout, or cryopin-associated periodic syndromes (CAPS).

40. 27. The use of claim 26, wherein the polypeptide comprises glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), oxyntomodulin, GLP-1 receptor binding polypeptide, GIP receptor binding polypeptide or glucagon receptor binding polypeptide, and the disorder comprises diabetes, obesity, or metabolic syndrome.

41. 27. The use of claim 26, wherein the polypeptide comprises glucagon-like peptide-1 (GLP-1), dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, glucose-dependent insulinotropic peptide (GIP), oxyntomodulin, GLP-1 receptor binding polypeptide, GIP receptor binding polypeptide or glucagon receptor binding polypeptide, and the disorder comprises Parkinson's disease.

42. The use of claim 21 , wherein the recombinant expression vector comprises an adeno-associated virus vector.

43. 22. The use according to claim 21 , wherein the suitable regulatory sequences comprise a CMV promoter or the insulin-specific promoters RIP1 or RIP2.

44. The use of claim 21, wherein the genetically modified pancreatic islet organoid is surgically transplanted into the anterior chamber of the eye or the brain of a subject.

45. 1. A process for producing a genetically modified pancreatic islet organoid comprising pancreatic islet cells, comprising: (a) introducing a recombinant expression vector into a pancreatic islet cell, the recombinant expression vector comprising: (i) a nucleic acid sequence encoding a polypeptide that is not endogenously expressed in pancreatic islet cells; and (ii) suitable control sequences operably linked to said nucleic acid sequence; and (b) culturing the islet cells containing the recombinant expression vector in vitro to form the genetically modified islet organoids containing the islet cells. The process includes:

46. The introducing step (a) further comprises: (a1) preparing an aqueous mixture of said pancreatic islet cells containing said recombinant expression vector with a silk protein capable of assembling into water-insoluble macrostructures, optionally further comprising laminin; (a2) allowing the silk protein to assemble into water-insoluble macrostructures in the presence of the pancreatic islet cells containing the recombinant expression vector, thereby forming a 3D silk matrix of the pancreatic islet cells containing the recombinant expression vector. Including, The culturing step (b) comprises culturing the islet cells comprising the recombinant expression vector in vitro within the silk matrix to form the genetically modified islet organoids comprising the islet cells.

46. ​​The process of claim 45.

47. moreover: (c) disposing said genetically modified pancreatic islet organoids comprising pancreatic islet cells onto a 3D silk matrix consisting of a water-insoluble macrostructure of silk protein, and optionally further comprising laminin; and (d) allowing the genetically modified pancreatic islet organoids comprising pancreatic islet cells to attach to the 3D silk matrix.

47. The process of any one of claims 45 to 46, comprising:

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