STATEMENT OF RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/353,731, filed June 20, 2022, the entire contents of which are incorporated herein by reference for all purposes.

FIELD

Provided herein are compositions and methods for treating diabetes (e.g., Type I diabetes and insulin dependent type 11 diabetes). In particular, provided herein are insulin secreting cells and uses thereof in the treatment of diabetes.

BACKGROUND

Diabetes Mellitus (DM) is a group of common metabolic disorders associated with hyperglycemia. Based on the recent statistics from CDC, 37.3 million Americans (11.3% of the US population) have DM. Moreover, 96 million Americans are prediabetic. Similarly, several pets suffer from diabetes and insulin is prescribed to treat the diabetes.

Unfortunately, there is no curative treatment for DM in humans or pets, and the chronic management cost, in humans, for 2017 was $327 billion in direct medical costs and $90 billion in reduced productivity' (American Diabetes A. Economic Costs of Diabetes in the U.S. in 2017. Diabetes Care. 2018;41(5):917-28. Epub 2018/03/24. doi: 10.2337/dcil8- 0007). The major cause of pathological manifestations of DM is hyperglycemia, and insulin therapy is required at some point for both Type 1 and Type 2 DM. Thus, there is a huge population of Diabetics who are dependent on insulin to control their blood sugar. In recent years, there has been an astronomical increase in the cost of insulin and a significant outcry to reduce the cost of insulin (Rajkumar SV. The High Cost of Insulin in the United States: An Urgent Call to Action. Mayo Clin Proc. 2020;95(l):22-8. Epub 2020/01/07. doi: 10.1016/j.mayocp.2019.11.013; Conner F, Pfiester E, Elliott J, Slama-Chaudhry A. Unaffordable insulin: patients pay the price. Lancet Diabetes Endocrinol. 2019;7(10):748. Epub 2019/09/20. doi: 10.1016/82213-8587(19)30260-8).

With the recent advances in stem cell biology, there have been several studies examining the conversion of stem cells to insulm-producing cells (1PC) (Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV. Islet transplantation in seven patients with ty pe 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. The New England journal of medicine. 2000;343(4):230-8. Epub 2000/07/27. doi: 10.1056/NEJM200007273430401; Andres A, Kin T, O'Gorman D, Livingstone S, Bigam D, Kneteman N, Senior P, Shapiro AM. Clinical islet isolation and transplantation outcomes with deceased cardiac death donors are similar to neurological determination of death donors. Transpl Int. 2016;29(l):34-40. Epub 2015/08/13. doi: 10.1111/tri.12650; Markmann JF, Deng S, Desai NM, Huang X, Velidedeoglu E, Frank A, Liu C, Brayman KL, Lian MM, Wolf B, Bell E, Vitamaniuk M, DolibaN, Matschinsky F, Markmann E, Barker CF, Naji A. The use of non-heart-beating donors for isolated pancreatic islet transplantation. Transplantation.

2003;75(9): 1423-9. Epub 2003/06/07. dot: 10.1097/01. TP.0000061119.32575. F4). However, most of the studies failed or showed temporary improvement following allografted cadaveric islets and cord-blood stem cells converted IPC as a result of the graft versus host diseases (Chen S, Du K, Zou C. Current progress in stem cell therapy for type 1 diabetes mellitus. Stem Cell Res Ther. 2020;l l(l):275. Epub 2020/07/10. doi: 10.1186/sl3287-020-01793-6; Cheng SK, Park EY, Pehar A, Rooney AC, Gallicano GI. Current progress of human trials using stem cell therapy as a treatment for diabetes mellitus. Am J Stem Cells. 2016;5(3):74- 86. Epub 2016/11/18).

However, current approaches suffer from immune rejection, differentiation into other cell types that can lead to cancerous growth, continued proliferation, which can lead to significant hypoglycemia, and are time-consuming to produce.

Improved insulin-producing cells are needed.

SUMMARY

The compositions and methods described herein overcome limitations of previous insulin producing cells by using preadipocytes from the same subject, which can be autografted. Because the cells are isolated from a small amount of fat (50 mg) from the same organism, the chance of immune rejection is nonexistent. These preadipocytes undergo terminal differentiation into mature adipocytes, which are harmless. Also, a Pl 6 or other cell cycle inhibitor gene expressing cassette, which prevents any further cell division and a suicide gene, which will kill the cell if needed, provide further safety features. Overall, the cells are non-immunogenic, non-proliferating, and safe.

Accordingly, in some embodiments, provided herein is an isolated preadipocyte, comprising an exogenous insulin gene and one or more copy of a cycle inhibitor gene (e.g. P16 gene) cassete and a suicide gene (e.g., nitroreductase (NTR)). In some embodiments, the insulin gene, cell cycle inhibitor gene, and suicide gene are on one or more vectors. In some embodiments, the genes are operably linked to a promoter (e.g., a constitutive promoter or a glucose-responsive promoter (e.g., comprising one or more glucose response elements)).

Also provided is a composition comprising the preadipocytes described herein. In some embodiments, the composition is a hydrogel (e.g., Matrigel, alginate, or a PGLA-PEG- PGLALaponite hydrogel, etc.).

Further embodiments provide a method of producing insulin in a subject, comprising: a) isolating preadipocytes from the subject; b) engineering the preadipocytes to comprise an exogenous insulin gene with or without a cell cycle inhibitor gene, and with or without a suicide gene to generate engineered insulin secreting preadipocytes; and c) transplanting the engineered preadipocytes into the subject (e.g., subcutaneously or in the omentum). In some embodiments, the preadipocytes are isolated from subcutaneous fat of the subject. In some embodiments, the subject has Type 1 or 2 diabetes (e.g., or insulin-dependent Type II diabetes).

Certain embodiments provide a method of producing insulin and/or treating Type 1 and Type 2 diabetes in a subject, comprising, transplanting a composition described herein into the subject.

Other embodiments provide the use of a composition described herein to produce insulin in a subject and/or treat type I and type 2 diabetes in a subject.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

Fig. 1 shows panel A - isolated preadipocy tes from mice. Panel B - differentiation of preadipocytes into mature adipose tissue.

Fig. 2 shows panel A - construct. Panel B - luciferase production in proportion to cell number in mouse, human, and sheep preadipocy tes. N = 5.

Fig. 3 demonstrates insulin production in proportion to preadipocytes cells. N = 5.

Fig. 4 demonstrates blood glucose levels in animals comprising the cells described herein.

DEFINITIONS To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following tenns take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and/or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.”

As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent described herein (e.g., composition described herein) to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.

The term “administration” and variants thereof (e.g., “administering” a composition) in reference to cells or a compound means providing the cells or compound or a prodrug of the compound to the individual in need of treatment or prophylaxis When cells or a compound of the technology or a prodrug thereof is provided in combination with one or more other active agents, “administration” and its variants are each understood to include provision of the compound or prodrug and other agents at the same time or at different times. When the agents of a combination are administered at the same time, they can be administered together in a single composition, or they can be administered separately. As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combining the specified ingredients in the specified amounts. By “pharmaceutically acceptable” is meant that the ingredients of the pharmaceutical composition are compatible with each other and not deleterious to the recipient thereof.

The term “subject” as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation, or experiment.

The term “effective amount” as used herein means that amount of an agent (e.g., composition described herein) that elicits the biological or medicinal response in a cell, tissue, organ, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician. In some embodiments, the effective amount is a “therapeutically effective amount” for the alleviation of the symptoms of the disease or condition being treated. In some embodiments, the effective amount is a “prophylactically effective amount” for prophylaxis of the symptoms of the disease or condition being prevented.

A cell is said to be “genetically altered” when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. The polynucleotide will often comprise a sequence encoding a protein of interest, which enables the cell to express the protein at an elevated level. The genetic alteration is said to be “inheritable” if progeny of the altered cell has the same alteration.

DETAILED DESCRIPTION

Provided herein are compositions and methods for treating Type I and Type II diabetes. In particular, provided herein are insulin secreting cells and uses thereof in the treatment of Type I and Type II diabetes.

The compositions and methods described herein overcome limitations of previous insulin producing cells by using preadipocytes from the same subject, which can be autografted. Because the cells are isolated from a small amount of fat (50 mg) from the same organism, the chance of immune rejection is nonexistent. These preadipocytes undergo terminal differentiation into mature adipocytes, which are harmless. Also, a cell cycle inhibitor gene (e.g., P16 expressing cassette), which prevents any further cell division and a suicide gene, which will kill the cell if needed, provide further safety features. Overall, the cells are non-immunogenic, non-proliferating, and safe.

For example, in some embodiments, provided herein is an isolated preadipocyte engineered to comprise an insulin gene (e.g., human insulin gene). Insulin is synthesized as an inactive precursor molecule, a 110 amino acid-long protein called "preproinsulin". Preproinsulin is translated directly into the rough endoplasmic reticulum (RER), where its signal peptide is removed by signal peptidase to form "proinsulin". As the proinsulin folds, opposite ends of the protein, called the "A-chain" and the "B-chain", are fused together with three disulfide bonds. Folded proinsulin then transits through the Golgi apparatus and is packaged into specialized secretory vesicles. In the granule, proinsulin is cleaved by proprotein convertase 1/3 and proprotein convertase 2, removing the middle part of the protein, called the "C-peptide". Finally, carboxypeptidase E removes two pairs of amino acids from the protein's ends, resulting in active insulin - the insulin A- and B- chains, now connected with two disulfide bonds.

In some embodiments, the preadipocyte comprises a p!6 gene cassette and/or a suicide gene (e.g., nitroreductase (NTR)). pl6 (also known as p!6INK4a, cyclin-dependent kinase inhibitor 2A, CDKN2A, multiple tumor suppressor 1 and numerous other synonyms), is a protein that slows cell division by slowing the progression of the cell cycle from the G1 phase to the S phase, thereby acting as a tumor suppressor.

In some embodiments, the insulin gene, pl 6 gene, and suicide gene are on one or more vectors. In some embodiments, the genes are operably linked to a promoter (e.g., a constitutive promoter or a glucose-responsive promoter (e.g., comprising one or more glucose response elements)).

In some embodiments, the engineered preadipocytes described herein are encapsulated in a matrix (e.g., hydrogel). Examples include but are not limited to Matrigel or a PGLA-PEG-PGLA-Laponite hydrogel.

Additional encapsulation methods are specifically contemplated, in some embodiments, cells or cell clusters are encapsulated for transplantation into a subject. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles, and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, herein incorporated by reference in its entirety).

Methods of preparing microcapsules include those disclosed by Lu M Z, et al.

Biotechnol Bioeng. 2000, 70: 479-83; Chang T M and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu M Z, et al., J. Microencapsul. 2000, 17: 245-51.; herein incorporated by reference in their entireties. For example, microcapsules may be prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 pm. Such microcapsules can be further encapsulated with additional 2-5 pm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; herein incorporated by reference in its entirety). In some embodiments, microcapsules are based on alginate, a marine polysaccharide (Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; herein incorporated by reference in its entirety) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guamdine) hydrochloride in the presence of calcium chloride.

In some embodiments, cells are microencapsulated for transplantation into a subject (e.g., to prevent immune destruction of the cells). Microencapsulation of cells provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs). In some embodiments, cells and/or cell clusters are microencapsulated in a polymeric, hydrogel, or other suitable material, including but not limited to: poly(orthoesters), poly(anhydrides), poly(phosphoesters), poly(phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly(L-lysine), poly(gly colic acid), poly(lactic-co-gly colic acid), poly(lactic acid-co-lysine), poly(lactic acid- graft-lysine), polyanhydrides, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly(anhydride-co-imides), poly(amides), poly(ortho esters), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate), poly(caprolactone), poly(carbonates), poly(amino acids), poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes), poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, polystyrene, polysaccharides, alginate, hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIP A), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc., and their derivatives, co-polymers, and mixtures thereof. In some embodiments, cell are microencapsulated in an encapsulant comprising or consisting of alginate. Cells may be embedded in a material or within a particle (e.g., nanoparticle, microparticle, etc.) or other structure (e.g., matrix, nanotube, vesicle, globule, etc.). In some embodiments, microencapsulating structures are modified with immune- modulating or immunosuppressive compounds to reduce or prevent immune response to encapsulated cells. For example, pancreatic lineage cells are encapsulated within an encapsulant material (e.g., alginate hydrogel) that has been modified by attachment of an immune-modulating agent (e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1). In some embodiments, such an immune modulating agent is a T-cell chemorepellent and/or a pro-survival factor.

In some embodiments, cells are macroencapsulated for transplantation into a subject. Macroencapsulation of cells, for example, within a permeable or semi-permeable chamber, provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs), prevents spread of cells to other tissues or areas of the body, and/or allows for efficient removal of cells. Suitable devices for macroencapsulation include those described in, for example, U.S. Pat. No. 5,914,262; Uludag, et al., Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, herein incorporated by reference in their entireties.

Other encapsulation (micro or macro) devices and methods may find use in embodiments described herein. For example, methods and devices described in U.S. Pub No. 20130209421, U.S. Pat. No. 8,785,185, each of which are herein incorporated by reference in their entireties, are within the scope of embodiments described herein.

The engineered preadipocytes described herein find use in therapeutic approaches, including, but not limited to, transplantation of the cells into a subject. In practice, embodiments of the present disclosure provide a method of treating Type I diabetes using the cells described herein. In some embodiments, the method comprises the steps of a) isolating preadipocytes from a subject; b) engineering the preadipocytes to comprise an exogenous insulin gene, a P16 gene, and a suicide gene to generate engineered preadipocytes; and c) transplanting the engineered preadipocytes into the subject. In some embodiments, the preadipocytes are isolated from subcutaneous fat of the subject.

Cells may be implanted into an appropriate site in a recipient. Suitable implantation sites may include, for example, the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach, or a subcutaneous pocket.

In some embodiments, cells are used in drug testing applications. For example, in some embodiments, drugs or biological agents are tested. Indications for drug testing include any compound or biological agent in the pharmaceutical discovery and development stages, or drugs approved by drug regulatory agencies, like the US Federal Drug Agency. In some embodiments, drug testing applications determine the effects of new chemical entities on insulin production.

Embodiments of the present disclosure provide kits comprising the cells described herein. For example, in some embodiments, kits comprise cells (e.g., insulin-producing cells described herein). In some embodiments, kits further comprise reagents for use of cells (e.g., buffers, test compounds, controls, etc.).

EXAMPLES

Unless specified otherwise, the following experimental techniques were used in the Examples.

Example 1

Preadipocytes were generated from 50 mg of subcutaneous adipose tissue from mice. Culturing them in clusters leads to differentiation into mature adipocytes (Figure 1). This data indicates that cells will differentiate into adipocytes if implanted subcutaneously or in the omentum. Thus, there is a negligible chance of these cells forming teratomas, which were observed in the stem cell implantation (Prokhorova TA, Harkness LM, Frandsen U, Ditzel N, Schroder HD, Bums JS, Kassem M. Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel. Stem Cells Dev. 2009;18(l):47-54. Epub 2008/04/09. doi: 10.1089/scd.2007.0266). This approach provides a safe and better alternative to IPCs obtained from stem cells differentiation, where the efficiency is low and includes a significant number of unconverted stem cells.

Mouse and human insulin gene lentiviral vectors were used to transduce preadipocytes from mice, humans, and sheep to generate stable IPCs. The same vector also had a blasticidin gene for positive selection. A 100% IPC population was obtained in 1 week. In addition, a luciferase reporter enzyme was inserted at the end of insulin c-peptide with proper cleavage sites for easy detection of insulin. Results demonstrated a dose-dependent functional luciferase production (Figure 2) in preadipocytes from mice, humans, and sheep.

To demonstrate functional insulin protein formation and secretion by transduced preadipocytes, an ELISA was conducted on cell culture supernatant using a mouse insulin ELISA kit (Alpco Inc. Cat # 80-INSMS-E01). As shown in figure 3, 1.2-5 pIU of insulin secretion per cell/day was observed. Studies have demonstrated that 0.25 1U of insulin is required in mice (13). Thus, 50,000 cells should be sufficient to produce the insulin necessary in the mouse. On the other hand, humans make about 12 - 17 IU of insulin/day, which requires about 600,000 - 1 million total cell implantation. Similar is the case with other large animals.

To examine the in-vivo insulin synthesis and reduction of hyperglycemia, hyperglycemic mice were generated by injecting 180 mg/kg of streptozotocin (Sigma Aldrich Inc) by IP route. A single dose of streptozotocin is well known to ablate beta cells and is used to create an animal model for type I DM (14). As shown in figure 4, significant hyperglycemia (> 600 mg/dl; reported as “High” by ReliOne glucometer) was observed in mice on day 2 following streptozotocin injection. On day 3, after confirming sustained hyperglycemia, 100,000 1PC diluted in 50 ul Matngel were implanted in the intraperitoneal region. On day 5, a significant reduction in hyperglycemia was observed in the mice transplanted with IPC.

Example 2

Improving insulin-producing cells for in-vivo implantation. Replication-deficient IPC with a “kill switch”

The cell cycle is tightly controlled by cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Among various proteins, pl6 is a potent regulator of CDK4, and when expressed, it halts the progression of cells from G1 to the S phase. Studies have shown that the expression of pl 6 stops cell replication even in cancer cells (Frost SJ, Simpson DJ, Clayton RN, Farrell WE. Transfection of an inducible p!6/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line. Mol Endocnnol. 1999;13(11): 1801-10. Epub 1999/11/07. doi: 10. 1210/mend.l3. 11.0374). Thus, pl 6 genes are inserted in IPC generated from preadipocytes.

Furthermore, to increase the safety of the IPC, a suicide gene therapy approach is used via insertion of a “kill switch” in these cells. In a Phase-I human clinical trial, bacterial nitroreductase (NTR) has been demonstrated to be safe, and efficient ablation of the targeted cells was observed using a prodrug CB1954 (Searle PF, Chen MJ, Hu L, Race PR, Lovering AL, Grove JI, Guise C, Jaberipour M, James ND, Mautner V, Young LS, Kerr DJ, Mountain A, White SA, Hyde El. Nitroreductase: a prodrug-activating enzyme for cancer gene therapy. Clin Exp Pharmacol Physiol. 2004;31(l 1):811-6. Epub 2004/11/30. doi: 10. 1111/j.1440- 1681.2004.04085.x). This approach is used to insert an NTR gene along with pl6. To positively select cells which only have pl6 and NTR, a puromycin cassette is inserted in the same vector using a CMV promoter with p!6, NTR, and puromycin in a polycistronic lentiviral vector with 2A peptides for efficient cleavage (Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Vaseghi HR, Qian L, Liu J. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific reports. 2017;7(l):2193. Epub 2017/05/21. doi: 10.1038/s41598-017-02460-2). Experiments are conducted to examine continuous and stable insulin synthesis and cell proliferation. It is contemplated that IPC with pl 6 will not proliferate and will be able to produce a steady amount of insulin. These cells are transplanted into mice and examined for stability and targeted ablation. Thus, in the future, if there is significant hypoglycemia following implantation, targeted cell ablation can be achieved by administering CB1954. This provides an added layer of security.

Synthetic hydrogel nanoparticles.

Currently, Matrigel (Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10(9): 1886-90. Epub 2010/02/18. doi: 10.1002/pmic.200900758) is used to implant the IPCs in the omentum. Additional synthetic, bioinert, thermosensitive, and biodegradable substances to graft the IPC are examined. A recent study demonstrated that PGLA-PEG-PGLA Laponite hydrogel has all the above properties and is free of any cytotoxic or inflammatory effect. In addition, the addition of desferrioxamine (DFO) to this hydrogel stimulated a robust angiogenic response and penetration of the host blood vessel (Ono K, Sumiya M, Yoshinobu N, Dode T, Katayama T, UedaN, Nagahama K. Angiogenesis Promotion by Combined Administration of DFO and Vein Endothelial Cells Using Injectable, Biodegradable, Nanocomposite Hydrogel Scaffolds. ACS Appl Bio Mater. 2022;5(2):471-82. Epub 2022/01/21. doi: 10.1021/acsabm.lc00870) Alternatively, several synthetic, biodegradable, thermosensitive, angiogenic hydrogels are available for cell transplantation (Giraudo MV, Di Francesco D, Catoira MC, Cotella D, Fusaro L, Boccafoschi F. Angiogenic Potential in Biological Hydrogels. Biomedicines. 2020;8(10). Epub 2020/10/24. doi: 0.3390/biomedicines8100436).

Example 3

Ovine clinical study

Diabetic sheep are generated by injecting streptozotocin as published (Higdon HL, 3rd, Parnell PG, Hill JE, Spitzer JC. Streptozocin-induced pancreatic islet destruction in beef cows. Vet Pathol. 2001;38(6):715-20. Epub 2001/12/06. doi: 10.I354/vp.38-6-7I5; Alexander DP, Briton HG, Cohen NM, Mashiter K, Nixon DA, Smith FG, Jr. Streptozotocin induced diabetes in the newborn lamb. Biol Neonate. 1971 ;17(5):381-93. Epub 1971/01/01. doi: 10.1159/000240330; Dickinson JE, Meyer BA, Chmielowiec S, Palmer SM. Streptozocin-induced diabetes mellitus in the pregnant ewe. Am J Obstet Gynecol.

1991;165(6 Pt l): 1673-7. Epub 1991/12/01. doi: 10.1016/0002- 9378(91)90013-h). Similar to mice, sheep develop diabetes with the administration of streptozotocin (Alexander et al., supra; Dickinson et al., supra). Preadipocytes are isolated from 50 mg of subcutaneous fat in 1 week for IPC generation (Figure 2). The isolated preadipocytes are transduced with an insulin gene lentiviral vector to create stable primary cells. Basal insulin production is determined by ELISA. Next, cells are transduced with a pl6-NTR-puromycin lentiviral vector.

Basal blood glucose levels are determined in sheep using the ABL800 Flex blood gas analyzer every morning for 7 days. Once a stable blood glucose profile is established, streptozotocin is administered to 8 sheep to ablate pancreatic beta cells and establish type 1 DM in the sheep (Alexander et al., supra; Dickinson et al., supra). The remaining 4 sheep are used as a negative control. The average plasma glucose in sheep is ~55 mg/dl (Sasaki Y, Takahashi H. Insulin secretion in sheep exposed to cold. J Physiol. 1980;306:323-35. Epub 1980/09/01. doi: 10.1113/jphysiol.1980. sp013399). When the fasting blood sugar has crossed 150 mg/dl in the streptozocin administered sheep, IPC are implanted in 4 sheep omentum following the standard surgical procedure. The other 4 sheep with hyperglycemia are used as control and injected with IPC hydrogel (vehicle for IPC), and 4 normoglycemic sheep are administered HBSS (vehicle for streptozotocin) and hydrogel (vehicle for IPC). The sheep are for 6 months to determine the long-term effect of the implanted IPC.

A dose-response study is conducted using a suboptimal, optimal, and excessive number of cells to further characterize implanted IPC's efficacy, safety, and toxicity profile. Implanted sheep are kept for 6 months to determine the long-term efficacy, safety, and toxicity of the optimal dose of implanted cells.

All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.