Human cell and tissue expansion and methods of use for therapeutic purposes

Abstract

The present disclosure provides methods of amplifying tissue samples derived from humans for purposes of treating certain diseases. The method comprises: isolating a small amount of associated tissue by biopsy or other tissue acquisition methods; the tissue is amplified in vitro, and meanwhile, natural functions are reserved; and re-implanting the amplified tissue to alleviate human disease. One example includes ex vivo expansion of human pancreatic islet tissue and reimplantation of the expanded cells into an individual with Type 1 diabetes for treatment and long term relief or cure of disease.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 436,546, filed December 31, 2022, which is incorporated by reference in its entirety. BACKGROUND

[0003] Certain cell types in the body are capable of sustained growth and division. These include the skin, various hematopoietic bone marrow precursor cells, and the intestinal epithelium. For example, the human erythroid lineage (composed primarily of red blood cells (RBCs) and the corresponding bone marrow that synthesizes RBCs) is estimated to produce more than 100 billion RBCs per day (>2 million cells per second; Sender and Milo, 2021).

[0004] In another aspect, certain tissues in the adult human body are composed of cells that are, in most cases, essentially excluded from the cell division cycle. This is because certain functional cell types in the human body require precise control of cell division in order to maintain the health and well-being of the organism. Endocrine cells are one such example. These cells, which secrete hormones into the bloodstream that are essential for normal physiologic homeostasis, are an example of cells whose division is highly restricted by the organism.

[0005] Hormone-secreting tissues must be carefully controlled in order to prevent overproduction of any given hormone. Many endocrine cells synthesize a given hormone both constitutively and on a pulsatile, reactive basis in response to changes in the physiology of the organism. When cells produce a given hormone constitutively, it is imperative to carefully control the total number of such cells in the organism to maintain the health of the organism. If the number of hormone-producing cells is overproduced due to insufficient control of cell division (e.g., due to a sporadic mutation), the sustained production of the hormone (such as thyroid hormone, adrenaline, or insulin) will result in an excess of the respective hormone. If left untreated, excess hormone production will result in serious illness and can be fatal.

[0006] Human diseases such as pheochromocytoma, pituitary tumors that secrete growth hormone, thyroid tumors that secrete T3 and T4, and pancreatic tumors that secrete insulin (called insulinomas) are representative examples of cell overgrowth, where the respective secreted hormone can induce morbidity or even death. For example, if left untreated, an insulinoma will cause excessive insulin secretion, leading to episodes of severe hypoglycemia (which can cause convulsions and death), even though these tumors are typically small, rarely metastasize, and can be easily cured by surgical resection (Kasper et al. 2015: Harrison’s Principles of Internal Medicine).

[0007] For the above reasons, long-lived organisms such as humans and other mammals maintain strict control over cell division for certain classes of cell types in the body. Human cells maintain multiple layers of cell division lock mechanisms to prevent certain cell types such as endocrine cells, neurons, and glial cells from accidentally overgrowing. These cell division locks (or CDLs) maintain cells in a G0 state and prevent them from entering the cell cycle. One class of CDLs includes certain tumor suppressor proteins, while another class includes certain metabolic regulatory proteins. Cell types with strong CDLs include neurons that release potent neurotransmitters such as dopamine, glial cells such as oligodendrocytes, Schwann cells that produce myelin sheaths that insulate and protect neurons, endocrine cells such as beta cells that secrete insulin and alpha cells that secrete glucagon, and so on. SUMMARY

[0008] Certain human diseases are characterized by the loss of specific cell types contained within important tissues or organs in the body. In some cases, in adult humans, the lost cells or tissues are CDL cells. Examples include type 1 diabetes, selected cases of type 2 diabetes, Parkinson’s disease, Wolfram syndrome, multiple sclerosis, and so on (Kasper et al., 2015; Gholamzad et al., 2019; Buzzard et al., 2017; Harris et al., 2020; Harris et al., 2020)

[0009] In some cases, it is desirable to treat the underlying disease with replacement of the lost tissue and associated cells, a process broadly referred to as regenerative medicine. However, a key challenge in doing so is that it is difficult to stimulate many tissues of the body (and the cell types contained within these tissues) to grow in vivo (in the body) or ex vivo (outside the body).

[0010] Skin is an organ that can be effectively propagated outside the body, and significant treatments have been achieved in human skin patients such as burn victims and patients with skin conditions that have rare, life-threatening genetic mutations in skin cell development. In one case study, a 7-year-old patient with life-threatening junctional epidermolysis bullosa was effectively treated by transducing human primary keratinocytes derived from the patient with a Moloney murine leukemia virus promoter construct with the full wild-type version of the human LAMB3 cDNA (Hirsch et al., 2017). While it is true that human keratinocytes are readily cultured ex vivo, this is still a significant advance.

[0011] Other cell types are more difficult to culture ex vivo, such as cardiac muscle cells. This has prompted researchers to turn to stem cell-based approaches. The pioneering work of Yamanaka and colleagues in Japan showed that mature differentiated cells can be reprogrammed to an embryonic-like state and then differentiated into a variety of mature cell types with selected properties similar to their mature wild-type counterparts (Takahashi and Yamanaka, 2006; Yamanaka, 2020). The field of induced pluripotent stem cells (iPSCs) has seen a great deal of research over the past two decades, with mixed results in attempts to harness the potential of regenerative medicine, as described below. It has become clear that the process of reprogramming stem cells appears to resemble developmental pathways that occur during human embryogenesis, but there are key differences that are not yet understood that can impact the long-term safety and effectiveness of therapeutic programs based on their use.

[0012] For example, to generate sufficient numbers of cardiac muscle cells (myocytes) for the purpose of replacing damaged heart tissue caused by a heart attack, researchers turned to stem cell-based approaches to generate sufficient numbers of myocyte-like cells. iPSCs were used to generate large numbers of myocyte-like cells, which were then used to attempt to repair scar tissue formation induced by hypoxia, a consequence of myocardial infarction. The generated stem cell-derived myocytes had difficulty functioning normally when reintroduced into the heart of a primate. The stem cell-derived cardiac muscle cells did not contract in unison with existing cardiac muscle cells and were unable to adapt to normal electrical rhythms. Abnormalities in the electrocardiogram (ECG) of the reimplanted primate were evident, which led to the development of life-threatening arrhythmias (Eb ioMedicine, 2018).

[0013] Both embryonic stem cells and induced pluripotent stem cells can be differentiated into multiple cell types that appear to be identical to corresponding fully differentiated human cell types. When gene expression analysis is performed using fully differentiated adult cell-derived cells originating from specific tissues, such as cardiac muscle cells or pancreatic insulin-producing beta cells, and similar cell types are produced from laboratory-differentiated stem cells, many gene expression differences become apparent (Yamanaka, S., 2020). This raises questions about the long-term safety of stem cell-based therapies that are intended to be reintroduced into humans for therapeutic purposes (Kushner et al., 2014). In fact, there have been many cases of tumors arising after the introduction of stem cell-derived tissues into experimental animals. Teratomas, which are aggressive undifferentiated tumors, have been shown to form in all three major embryonic tissue layers (ectoderm, mesoderm, and endoderm) in mammals. This represents a serious long-term safety risk associated with the potential clinical use of iPSCs in humans (Blum et al., 2009; Cunningham et al. 2012; Fong et al., 2010; Kum et al., 2012; Lee et al., 2009; Mutter et al., 1987; Prokhorova et al., 2009).

[0014] If there were a technology that could induce a cell division lock mechanism that controls the release of certain adult tissues and cell types, such as human beta cells, from the cell division lock that prevents them from growing and dividing in large numbers, the aforementioned problems and safety issues associated with the use of stem cells could be avoided. The present disclosure provides for the first time the ability to select a therapeutically relevant, fully differentiated, non-dividing cell or cell population and temporarily release them from the cell division lock in order to allow a controlled cell division for a period of time until a therapeutically sufficient amount of cells or tissue has been produced. This is achieved by inducing a temporary homeostatic thermodynamic shift in the energy utilization within the cell, thereby enabling the cell to switch to the cell cycle without resorting to the use of recombinant DNA methods or technologies, such as CRISPR / CAS-9 genome editing methods. In order to maintain the genomic integrity of otherwise healthy human cell types that are intended for re-implantation and only lack relative abundance in the body, the use of the latter should be avoided. Therefore, for the purpose of patient safety, the use of recombinant DNA and genome editing methods is preferably avoided, and the temporary modulation of RNA expression levels by the use of gene silencing methods, such as small interfering RNA (siRNA), is useful and a preferred embodiment.

[0015] The essence of the present disclosure relates to the normal regulation of cell divisional lock that temporarily releases the ability of certain human tissues and cell types to grow ex vivo. By temporarily removing the cell divisional lock (CDL) that prevents entry into the cell cycle of vital tissue-specific cells, such as endocrine cells or neurons, in a controlled, reversible manner, the techniques described herein make it possible to expand such cell types ex vivo while maintaining their original phenotypic characteristics as mature, fully differentiated cells. This approach is fundamentally different from existing approaches that involve the use of embryonic stem cells (ES cells) or induced pluripotent stem cells (iPSCs), in which cells are first grown in a dedifferentiated state to sufficient numbers, and then subsequently redifferentiated into a cell type with properties close to the cell type required for therapy. Figure 1 A diagram is provided that outlines the differences between current approaches to stem cell-based therapies and the disclosure provided herein.

[0016] Accordingly, the present disclosure enables skilled researchers to expand selected cell types ex vivo to therapeutic quantities for future reimplantation of the cell types into humans while avoiding the need to use stem cell-based approaches to obtain sufficient cell growth. This approach avoids the safety, genetic expression variability, and tumorigenicity issues associated with stem cell use (Ohnishi et al., 2014; Yasuda et al., 2018).

[0017] Unless otherwise indicated, the terms used herein are to be understood as taking the meanings commonly used in the relevant art. Several terms used herein and their meanings are shown below.

[0018] As used herein, the term “tissue” refers to a group or layer of similar specialized cells that work together to perform some special function.

[0019] As used herein, the terms “chemical agent” and “biological agent” are used interchangeably and both refer to chemical and biological pharmaceutical agents.

[0020] As used herein, the term “chemical agent” refers to a substance with a molecular weight of up to, but not including, 2000 atomic mass units (Daltons). Such substances are sometimes referred to as “small molecules”.

[0021] As used herein, “biological agents” refers to molecules that include proteins and polynucleotides and have a molecular weight equal to or greater than 2000 atomic mass units (“amu” or “Daltons”), but no more than 990,000 amu.

[0022] As used herein, the terms “activator” and “agonist” are used interchangeably.

[0023] As used herein, the terms “inhibitor” and “antagonist” are used interchangeably.

[0024] "Expression inhibitor" is intended to include a compound that inhibits the expression of a gene by any mechanism, including interfering with the production of functional mRNA or enhancing the degradation of mRNA.

[0025] For the present disclosure, the expression "a substance 'inhibits' means that the substance can bind to a protein present in a cell, tissue, blood, or in vivo and reduce its activity; the substance can reduce or eliminate the function of the protein; the substance can reduce the amount or level of the protein; and / or the substance can reduce the expression or production of the protein.

[0026] Unless specifically noted otherwise, "inhibitor" is synonymous with "antagonist." The inhibition by an inhibitor can be partial or complete. The terms "bind(s)," "binding," and "binds to" have their ordinary meaning in the field of biochemistry in describing the interaction between two substances (e.g., enzyme-substrate, protein-DNA, receptor-ligand, etc.). As used herein, the term "binds to" is synonymous with "interacts with" in the context of discussing the relationship between a substance and its corresponding target protein or nucleic acid.

[0027] As used herein, the term "antibody" refers to a protein or immunoglobulin produced in response to an antigen and can "specifically bind" to that antigen. An antibody that "specifically binds" to an antigen is one that interacts only with the epitope of the antigen that induced the synthesis of the antibody or with a structurally related epitope. An antibody that "specifically binds" to an epitope is able to interact with that epitope under appropriate conditions, even in the presence of multiple potential binding targets.

[0028] As used herein, the term "antigen" refers to a protein or peptide target having an epitope to which an antibody specifically binds.

[0029] As used herein, the term "fragment" refers to a portion of a polypeptide or polynucleotide. In one embodiment, the fragment retains the activity of the polypeptide or polynucleotide.

[0030] According to the present disclosure, a therapeutically effective amount of one or more compounds / substances that upregulate / activate or downregulate / inhibit, respectively, the function or expression level of, for example, a viability target (VT) protein or a protein that degrades a VT, is administered to a cell obtained from a mammal or a mammal in need thereof. The term "mammal" as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic animals, and farm animals.

[0031] As used herein, the phrase “effective amount” means an amount of compound, material, cell, cell group, or composition comprising the compounds disclosed herein that is capable of effectively producing some desired effect in at least one cell subpopulation of an animal (e.g., pancreatic cells), such as increasing the expression of a viable target, increasing the amount of a viable target, and / or reducing the activity of a protein or nucleic acid sequence that inhibits a viable target.

[0032] As used herein, the phrase “therapeutic effective amount” means the amount of a compound, material, cell, cell group, or composition comprising a compound of the present disclosure that effectively produces some desired therapeutic effect in at least one cell subpopulation in an animal with a reasonable benefit / risk ratio suitable for any medical treatment (e.g., reasonable side effects of administering any medical treatment).

[0033] Unless otherwise stated, “a / an”, “the” and “at least one” are used interchangeably and mean one or more.

[0034] Conditions that are “suitable” or “appropriate” for an event to occur are those that do not prevent such an event from happening. Therefore, these conditions allow, enhance, promote, and / or contribute to the event.

[0035] As used herein, in the context of a composition, cell, cell group, antibody, nucleic acid, or small molecule, “providing” means manufacturing, purchasing, or otherwise obtaining the composition, antibody, nucleic acid, or small molecule.

[0036] As used in this specification and the appended claims, the term "or" is generally used in its meaning as including "and / or" unless otherwise expressly stated. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. In some cases the use of "and / or" does not mean that in other cases the use of "or" may not mean "and / or".

[0037] The terms "preferred" and "ideally" refer to embodiments of this disclosure that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of this disclosure.

[0038] As used in this article, the terms "have", "has", "having", "include", "includes", "including", "comprise", "comprises", and "comprising" are used in an open and inclusive sense, and usually mean "including but not limited to", "including but not limited to", or "including but not limited to".

[0039] It should be understood that wherever embodiments are described herein using the terms “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising,” etc., similar embodiments in other respects described according to “consisting of” and / or “substantially consisting of” are also provided. The term “consisting of” means including and limited to anything following the phrase “consisting of.” That is, “consisting of” indicates that the listed element is necessary or mandatory, and that other elements may be absent. The term “substantially consisting of” means including any element listed following the phrase, and may also include other elements besides those listed, provided that such elements do not interfere with or affect the activity or function specified for the listed elements in this disclosure.

[0040] References to "an embodiment," "an embodiment," "some embodiments," or "a number of embodiments," etc., throughout this specification mean that a particular feature, construction, composition, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, such phrases appearing throughout the specification do not necessarily refer to the same embodiment of this disclosure. Furthermore, particular features, constructions, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0041] Throughout this disclosure, various aspects of this disclosure may be presented in a range format. It should be understood that the range format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this disclosure. Therefore, a description of a range should be considered as having specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered as having explicitly disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0042] In this specification, specific embodiments may be described individually for clarity. Unless otherwise expressly stated, features of a particular embodiment are incompatible with features of another embodiment; otherwise, some embodiments may include combinations of compatible features described herein that are associated with one or more embodiments.

[0043] For any method disclosed herein that includes discrete steps, these steps can be performed in any feasible order. Furthermore, any combination of two or more steps can be performed simultaneously, if appropriate. Attached Figure Description

[0044] The following detailed description of exemplary embodiments of this disclosure can be better understood when read in conjunction with the accompanying drawings.

[0045] Figure 1 A schematic diagram illustrates the differences between expanding cell clumps using stem cells and fully differentiated mature cells. The stem cell approach involves a three-step process: (1) dedifferentiating mature cells into induced pluripotent stem cells (iPSCs), thereby losing the mature cell phenotype; (2) allowing the iPSCs to proliferate until a sufficient number are reached; and (3) redifferentiating the iPSCs into structurally and functionally similar but not identical mature differentiated cells. This new approach involves preserving the fully differentiated phenotype and inducing cells to enter the cell cycle in a temporary and reversible manner.

[0046] Figure 2 This study demonstrates enhanced viability of 32D cells expressing insulin receptor and insulin receptor substrate 2 (IRS-2) after treatment with either insulin (INS) or the IRS activator (HP-508). 32D cells treated with the IRS activator... IR-IRS-2Cells exhibited substantially the same growth as INS-treated cells, but showed enhanced viability after 72 hours of culture compared to INS-treated cells (Housey and Balash, 2018; Housey and Balash, 2023). Note the presence of abundant cytoplasmic inclusions in INS-treated cells, reflecting increased cellular stress and decreased viability. In contrast, IRS-activated cells exhibited multiple intact cellular morphological regions (circled). INS-treated cells typically showed viability values ​​of 60% or lower, while IRS-activated cells could exhibit viability values ​​exceeding 90% after 72 hours (data not shown). Viability was determined using trypan blue exclusion assay and an automated cell counter.

[0047] Figure 3 shows the growth curves of human islet tissue maintained in ex vivo cultures in the presence of IRS activators and growth promoters. The growth of β-cell-containing human islet tissue in the presence of IRS-2 activator (HP-508) combined with repeated treatments with different siRNAs is shown in the figure. Tissue growth was determined by digital imaging using a Zeiss CD-7 automated microscope. Total tissue surface area was calculated using ZEN software. Under these conditions, human islet tissue less than 300 μm in length typically develops into tissue blocks with a vertical height (thickness) ranging from 15 to 30 μm. TP53, grew in the presence of siRNA inhibiting the expression of the human tumor suppressor protein P53 (see Figure 4); STK4, grew in the presence of siRNA inhibiting the expression of the human serine / threonine kinase 4 gene (see Figure 4); VGLL4, grew in the presence of siRNA inhibiting the expression of the human degenerative-like family member 4 gene (see Figure 4); TSC-1, grew in the presence of siRNA inhibiting the expression of the human tuberous sclerosis 1 gene (see Figure 4).

[0048] Figure 4 shows examples of synthesized and tested siRNAs. Each siRNA is shown in two formats. The top diagram shows each strand of the duplex from 5' to 3', and ribonucleotides with an "r" preceding nucleotides A, U, G, or C are labeled. Those nucleotides without an "r" preceding nucleotides A, T, G, or C represent deoxyribonucleotides. The bottom diagram shows the two strands of the duplex hybridization.

[0049] Figure 5 This demonstrates insulin production through the cultivation of human islets. The islets continuously produce insulin throughout their growth period.

[0050] Figure 6A representative human islet-like tissue mass, approximately 1,000 micrometers (μm) x 400 μm x 170 μm, was shown propagated from a small group of human donor cells (<10 cells) using an IRS activator. The representative human islet tissue mass shown was maintained in culture for more than one year. The abbreviations are as follows: it – islet tissue; v. – angiogenesis. Note that the length dimension of the tissue mass exceeds 1,000 micrometers. Angiogenesis-like vascularization is visible throughout the tissue mass, appearing in a penetrating manner. These structures appear during the mid- and late-stages of growth. As shown in Figure 3, the addition of a growth enhancer (also referred to herein as a cell division enhancer) can reduce the total growth time of a tissue mass sufficient to support reimplantation to only 120 to 180 days.

[0051] Diagrams are not necessarily drawn to scale. Similar numbers used in diagrams refer to similar components, steps, etc. However, it should be understood that using numbers to refer to components in a given diagram does not restrict components labeled with the same numbers in another diagram. Furthermore, using different numbers to refer to components does not mean that components with different numbers cannot be the same as or similar to components with other numbers. Detailed Implementation

[0052] This disclosure includes a method for in vitro expansion of cells derived from a patient. The method may include providing a tissue sample or similar specialized cells (target cells) from a subject, such as a tissue sample obtained by biopsy or an enriched or purified population of target cells. The tissue may include similar specialized cells that collectively perform a specific function. The tissue may originate from an individual suffering from a disease in which the pathology includes tissue loss. Examples of such diseases include, but are not limited to, type 1 diabetes (T1D) and Parkinson's disease. In one embodiment, the disease is T1D, and the subject has a relative or absolute deficiency of insulin. The similar specialized cells are the cells to be expanded in vitro. When the disease is T1D, examples of tissue and similar specialized cells include, but are not limited to, pancreatic tissue, islets, and β cells. When the disease is Parkinson's disease, examples of tissue and similar specialized cells include, but are not limited to, substantia nigra tissue and dopaminergic neurons.

[0053] Establishing tissues with the desired target cells in culture. In one embodiment, this may also include establishing cells (e.g., tissue samples and / or target cells) in culture, which can be done using known enriched culture media (such as Durbeco Modified Eagle Medium supplemented with 10% human serum). Other types of culture media can be used (Yao and Asayama, 2017). Higher percentages of serum can be used if necessary. If the relevant cell types are viable in in vitro culture, it is not necessary to identify viability targets or to determine compounds that can activate, stabilize, or reduce the degradation of viability targets. The terms viability and viability refer to maintaining the cell type in culture without significant loss of the total cell count, although net cell growth is not required. Figure 2 This illustrates a representative example of the effect of compound HP-508 on the viability of cultured human β cells.

[0054] Cell expansion (e.g., tissue samples and / or target cells) involves culturing in the presence of compounds that activate target proteins whose cellular function is essential for maintaining cell viability when the desired cell type is grown in laboratory culture. This target protein or nucleic acid sequence is called a viability target (“VT”). An example of a viability target for some cells is the IRS-2 protein. Cells expressing the IRS-2 protein include pancreatic islet cells (e.g., β cells) and substantia nigra (e.g., dopaminergic neurons). In other embodiments, viability targets for specific tissues or cells can be identified by selecting target proteins or nucleic acid sequences (such as miRNAs) from the human genome (e.g., the REF genbank). This can be achieved using a variety of standard methods, but one of the most common is through targeted gene knockout studies in mice or other primates. Most cell types do not require the use of viability targets because it has been well demonstrated that most human and other mammalian cell types remain viable in in vitro culture even without cell growth / expansion. In rare cases, similar to human β cells, transplanted pancreatic islet tissue containing live β cells cannot maintain viability indefinitely in culture. In such cases, suitable viability targets can be identified using a systematic gene knockout study, employing a single gene expressed in the target cell type. Gene expression analysis and the generation of knockout mice are standard procedures in the field. In these situations, the identification of suitable viability targets is determined by the loss of the corresponding target cell type during embryogenesis or in an early period after birth in the knockout mouse, as the relevant cell type is gradually lost due to the absence of the corresponding gene encoding the VT protein (Withers et al., 1998; Withers et al., 1999; Hennige et al., 2003). Example 1 illustrates this approach.

[0055] In these embodiments where the identification of viability targets is required, the method further includes selecting or designing compounds known to activate, stabilize, or reduce VT degradation. Such compounds are referred to herein as viability target enhancers (“VTEs”). If a compound is known to activate VT, it can be used. Alternatively, if such compounds are not available, they can be created, for example, by targeting genes known to cause VT degradation using antisense oligonucleotides or small interfering RNAs (“siRNAs”). The net effect of this treatment is an increase in the relative intracellular abundance of VT. VTEs that activate, stabilize, or reduce the degradation of the viability target IRS-2 protein include IRS activators. A non-limiting example of an IRS activator is HP-508 (Housey and Balash, 2018; Housey and Balash, 2023).

[0056] Cell expansion (e.g., tissue samples and / or target cells) involves culturing in the presence of a substance that enhances cell division. In one embodiment, this substance is one that reduces the expression of a cell division-locking (CDL) protein. Reducing CDL protein expression leads to the initiation and maintenance of cell growth. Using a cell division-enhancing substance minimizes the amount of time cells can be grown in vitro before reimplantation into a patient. Maintaining the shortest possible in vitro culture time helps maintain one or more phenotypic characteristics (hormone secretion, neurotransmitter release, etc.) of the selected cell type, which are essential for obtaining a therapeutically effective cell population for reimplantation. Suitable CDL proteins include, but are not limited to, proteins that control metabolic states and proteins that prevent tumor formation (tumor suppressor factors). Specific examples include, but are not limited to, synthesizing and testing siRNAs alone that enhance the growth of target cells, wherein specific siRNAs are designed to target proteins encoded by one of the following genes: activin gene (all variants), myosin (all variants), ANX7, APC, ARF, ATM, ATR, BCL2, BECLIN1, BIM, BLM, BMPR, BRCA1, BRCA2, BUB3, α-catenin, CBFA2 / AML1 / RUNX1, CDH1 (E-CAD; E-cadherin), CDKN1A, CDKN1B, CDKN2A, CDKN2C, CHK1, EXT1, EXT2, FBXW7 (CDC4), FEN1, FHIT, FST, H2AX, HIPK2, HRPT2, INPP4B, integrin, LIG4, LKB1, MAD2, MEN1, MEN2, MKNK2, MLH1, MSH2, MSH6, MUTYH, NBS1, NF1, NF2, NKX3.1 (NKX3A), P15, P53, PLK4, PMS1, PMS2, PTC, PTCH, PTEN, PTPN1, RB, RB1, RECQL4, ribosomal protein genes (L35, L37A, RPS19, S8), RNASEL, SDH, SMAD2, SMAD3, SMAD4 / DPC4, STK4, SU(FU), TEAD1, TGFH, TGFβR, TSC-1, TSC-2, VGLL4, VHL, WRN, WT1, and TP53. Specific examples include TP53, STK4, VGLL4, TSC-1, CDKN1B, and CDKN2A (Macleod, 2000; Payne and Kemp, 2005). Optionally, target cells can be used for screening to identify siRNA or RNA sequences or RNA analogs that can reduce CDL protein expression. This will temporarily re-enter the cell cycle for a sufficiently long period to expand the population and achieve therapeutically sufficient cell clumps.

[0057] Those skilled in the art will recognize that there are various methods known in the art for reducing the expression of CDL proteins. Such methods include, but are not limited to, the following.

[0058] Gene silencing using short interfering RNA (siRNA) and related methods using RNA interference (RNAi) are preferred embodiments (Housey and Balash, 2019). It is now clear that RNAi plays a crucial role in posttranscriptional gene silencing through molecules such as siRNA. siRNA is a double-stranded RNA (dsRNA) molecule of approximately 19 to 22 nucleotides (nt) that can reduce or silence messenger RNA (mRNA) translation in a sequence-specific manner (Walton et al., 2010; Sibley et al., 2010).

[0059] Polynucleotides can be used to reduce the expression of specific genes. Such repressive polynucleotides include RNA interference (RNAi) mediated by double-stranded small interfering RNA (siRNA), which can silence genes with high specificity. siRNA contains a sequence complementary to the messenger RNA (mRNA) encoding the protein and causes mRNA degradation. Those skilled in the art can design and synthesize siRNA molecules that reduce CDL protein expression. siRNA molecules for reducing CDL protein expression are also commercially available (e.g., Dharmacon, Lafayette, Colo.). Automated synthesis of nucleic acids is well-established and includes modifications at multiple sites in the nucleoside and ribose / deoxyribose ring systems (Sibley et al., 2010; Walton et al., 2010).

[0060] Another class of repressive nucleotides includes antisense RNA, a single-stranded RNA complementary to the protein-coding mRNA, which hybridizes with the mRNA and thus prevents its translation into a protein. The siRNA used in this method has the ability to reduce CDL protein expression. RNA interference methods represent a useful approach for molecularly targeted therapies. Therefore, in another embodiment, siRNA or another RNAi method is utilized. siRNA is synthesized and its ability to reduce CDL protein expression in a therapeutically effective manner in mammals is tested. The oligonucleotide synthesis method for preparing siRNA is well established. There are no limitations on the type of RNAi that can be used to reduce CDL protein levels in mammals, including RNA-DNA chimeras, tandem hairpin RNAs, tandem siRNAs, tRNA-shRNAs, etc. (Sibley et al., 2010).

[0061] In one embodiment, the polynucleotides available herein include double-stranded RNA (dsRNA) polynucleotides. The sequence of the polynucleotide comprises a single strand, referred herein as the sense strand, which is 16 to 30 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The sense strand is substantially identical, preferably identical, to the target mRNA (e.g., mRNA encoding a CDL protein). As used herein, the term “identical” means that the nucleotide sequence of the sense strand has the same nucleotide sequence as a portion of the target mRNA. As used herein, the term “substantially identical” means that the sequence of the sense strand differs from the sequence of the target mRNA by 1, 2, or 3 nucleotides (preferably 1 nucleotide), and the remaining nucleotides are identical to the sequence of the mRNA. These 1, 2, or 3 nucleotides of the sense strand are referred to as non-complementary nucleotides. When the polynucleotide contains a sense strand substantially identical to the target mRNA, these 1, 2, or 3 non-complementary nucleotides are preferably located in the middle of the sense strand. For example, if the sense strand is 21 nucleotides long, the non-complementary nucleotide is typically located at nucleotides 9, 10, 11, or 12, preferably at nucleotide 10 or 11. The other strand of the dsRNA polynucleotide (referred to herein as the antisense strand) is complementary to the sense strand.

[0062] The sense and antisense strands of dsRNA polynucleotides can also be covalently attached, typically via spacers composed of nucleotides. These polynucleotides are commonly referred to in the art as short hairpin RNAs (shRNAs). When the sense and antisense strands pair bases, the spacer region forms a loop. The number of nucleotides constituting the loop can vary, and loops ranging from 3 to 23 nucleotides have been reported (Sui et al., Proc. Nat'l. Acad. Sci. USA, 99, 5515-5520 (2002); and Jacque et al., Nature, 418, 435-438 (2002)).

[0063] In one embodiment, the polynucleotide useful herein includes a single-stranded RNA (ssRNA) polynucleotide. The sequence of the polynucleotide comprises a chain of at least 16 nucleotides, referred herein as the antisense strand. The antisense strand is substantially complementary, preferably complementary, to the target mRNA (e.g., mRNA encoding a CDL protein). In one embodiment, the polynucleotide used to reduce expression of a coding region in a cell comprises substantially all of the coding region, or in some cases, the entire coding region. The antisense strand is substantially complementary, preferably complementary, to the target coding region or the target mRNA. As used herein, the term "substantially complementary" means that at least one, two, or three nucleotides of the antisense strand are not complementary to the nucleotide sequence of the target mRNA.

[0064] The polynucleotides disclosed herein preferably possess biological activity. Biologically active polynucleotides induce post-transcriptional inhibition of expression of the target coding region, also known as silencing. Without being theoretically limited, upon introduction into cells, the polynucleotides of this invention hybridize with the target mRNA and signal an endonuclease to cleave the target mRNA. The result is inhibition of the expression of the polypeptide encoded by the mRNA. Whether the expression of the target coding region is inhibited can be determined, for example, by measuring a reduction in the amount of target mRNA in the cell, a reduction in the amount of the polypeptide encoded by the mRNA, or a decrease in the activity of the polypeptide encoded by the mRNA.

[0065] The polynucleotides disclosed herein may contain additional nucleotides. For example, for a sense strand, the 5' end, 3' end, or both ends may contain additional nucleotides, provided that these additional nucleotides are identical to the corresponding target mRNA and the total length of the sense strand is no more than 30 nucleotides.

[0066] Polynucleotides can be modified. Such modifications can be used to improve the stability of polynucleotides in certain environments. Modifications can include nucleic acid sugars, bases, or backbones, or any combination thereof. Modifications can be synthetic, naturally occurring, or non-natural. Polynucleotides can contain modifications at one or more nucleic acid sites present in the polynucleotide. Examples of backbone modifications include, but are not limited to, phosphonoacetate, thiophosphonoacetate, thiophosphate, dithiophosphate, aminophosphate, methyl phosphonate, chiral methyl phosphonate, 2-O-methylribonucleotides, and peptide nucleic acids. Examples of nucleic acid base modifications include, but are not limited to, modifications of inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), or 6-azpyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine), or propyne. Examples of nucleic acid sugar modifications include, but are not limited to, 2'-sugar modifications, such as 2'-O-methylnucleotide, 2'-deoxy-2'-fluoronucleotide, 2'-deoxy-2'-fluoroarabinose, 2'-O-methoxyethylnucleotide, 2'-O-trifluoromethylnucleotide, 2'-O-ethyl-trifluoromethoxynucleotide, 2'-O-difluoromethoxy-ethoxynucleotide, or 2'-deoxynucleotide. Polynucleotides containing such modifications can be obtained through commercial synthesis (e.g., Dharmacon Inc., Lafayette, Colo.).

[0067] Those skilled in the art will recognize that the method may include confirming whether the target tissue and / or cells maintain one or more appropriate phenotypic characteristics (hormone secretion, neurotransmitter release, etc.) of the selected cell type, which are essential for obtaining a therapeutically effective cell population for reimplantation. For example, when the target tissue and / or cells are derived from pancreatic tissue (e.g., islets of Langerhans), it is desirable to continuously produce insulin; and when the target tissue and / or cells are derived from substantia nigra (e.g., dopaminergic neurons), it is desirable to continuously produce dopamine.

[0068] Once a therapeutically effective quantity of cells / tissue is obtained, the cells are re-implanted into the appropriate site in the body. For example, in the case of β-cells used to treat diabetes, the cells can be directly infused into the liver via the portal vein (McEachron et al., 2018). In the case of dopamine-secreting neurons used to treat Parkinson's disease, standard neurosurgical procedures can be used to re-implant the cells into the substantia nigra via a real-time imaging-guided catheter, followed by infusion of the cells into an isotonic buffer solution. The number of re-implanted cells can vary depending on the patient's disease state and tissue loss. In some embodiments, at least 100,000 cells, at least 200,000 cells, at least 300,000 cells, at least 400,000 cells, at least 500,000 cells, or at least 600,000 cells are administered. In some embodiments, no more than 1,000,000,000,000 cells are administered.

[0069] The target tissue and / or cells can be expanded from their stock solution and cryopreserved for future use in cases where disease relapse occurs years or decades later and reimplantation of additional functional tissue would be effective. This method can achieve durable, long-term remission or functional cure for certain diseases, such as T1D and Parkinson's disease.

[0070] Optionally, the method may further include monitoring the status of the re-implanted tissue. When the re-implanted tissue contains β cells, the method may further include determining whether symptoms associated with T1D have been alleviated, such as whether the subject's glycemic control has been restored. When the re-implanted tissue contains dopaminergic neurons, the method may further include determining whether symptoms associated with Parkinson's disease have been alleviated, such as whether the subject's dopamine production has increased. Alternatively, elevated levels of brain-derived neurotrophic factor may also be monitored (see Example 2 below).

[0071] Type 1 diabetes (T1D, formerly known as juvenile diabetes) occurs when a patient's immune system mistakenly attacks a population of beta cells within the islets of Langerhans in the pancreas. Therefore, T1D is an autoimmune disease. Current standard care for T1D revolves around continuous daily insulin administration; several types of insulin have been developed and are available for use.

[0072] Various cellular approaches using islets derived from different sources (such as bovine, swine, and human cadavers) have been attempted to treat T1D, with limited success. The longest-lasting method is transplantation of human islets isolated from cadaveric donors. Because these donors and recipients are genetically different, recipients of allogeneic transplants must continuously take immunosuppressive drugs to reduce the likelihood of immune-mediated transplant rejection. Even if such transplants are successful, they typically only last for three to five years, after which the transplanted tissue ceases to function, and the recipient either resumes daily insulin use or must receive a second transplant from another allogeneic donor.

[0073] The serious, life-threatening limitations of allogeneic transplantation have led the FDA to classify all forms of allogeneic transplantation as experimental procedures. A detailed discussion of potential side effects and adverse events associated with allogeneic transplantation can be found on the National Institute of Diabetes, Digestive Disorders and Kidney Diseases (NIDDK) website (see Pancreatic Islet Transplantation: https: / / www.niddk.nih.gov / health-information / diabetes / overview / insulin-medicines-treatments / pancreatic-islet-transplantation). As described therein, the most serious side effects and adverse events associated with allogeneic transplantation are related to the need for long-term immunosuppression to avoid graft-versus-host disease in the recipient and immune rejection, which can lead to loss of function of the transplanted donor islets. Long-term immunosuppression is required even when the donor islets are derived from induced pluripotent stem cells rather than from a cadaveric pancreas. The recipient may develop antibodies against the donor cells, which can complicate the search for a suitable organ donor if another transplant is needed in the future (a common occurrence).

[0074] Most importantly, long-term use of immunosuppressants (essential to prevent the destruction of donor islets, whether derived from iPSCs or cadaveric donors) can lead to a number of serious side effects. According to the NIDDK website, potential side effects from long-term immunosuppressant use include: increased risk of infection, higher incidence of cancer, gastrointestinal side effects (such as vomiting, nausea, or diarrhea), headache, tremors, high blood pressure, high cholesterol and triglyceride levels, and kidney damage (https: / / www.niddk.nih.gov / health-information / diabetes / overview / insulin-medicines-treatments / pan creatic-islet-transplantation).

[0075] Despite the best efforts of many laboratories, it remains impossible to sustain human islet tissue isolated from cadaveric donors in vivo for more than a few months. During this period, the tissue does not exhibit significant growth, and insulin synthesis and release gradually decline. Therefore, cellular methods for replacing insulin-producing β-cells either rely on immediate allogeneic transplantation after isolation from cadaveric donors or on the use of iPSCs that differentiate into insulin-producing "β-cell-like" cells. Both methods still require the use of immunosuppressive drugs, and the iPSC method has the aforementioned additional long-term safety concerns.

[0076] Example

[0077] The following examples illustrate this disclosure. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly within the scope and spirit of this disclosure as set forth herein.

[0078] Example 1

[0079] Type 1 diabetes can be treated by generating β cells from isolated cloned human insulin.

[0080] It is well known that human islets containing β cells maintained in tissue or organ cultures cannot proliferate to a sufficient quantity for reimplantation surgery. Therefore, to maintain cultured cells without continuous cell loss, viability targets need to be identified and activated as described above. For β cells contained within the islets of Langerhans, mouse gene knockout experiments have demonstrated that insulin receptor substrate-2 (IRS-2) helps maintain viability (Hennige et al., 2003; Kuznetsova et al., 2016). Consequently, animals with both alleles of IRS-2 knocked out develop diabetes shortly after birth, while transgenic replacement of IRS-2 can cure diabetes. Therefore, IRS-2 meets the criteria for a viability target (VT).

[0081] These studies used a compound known to activate IRS-2, called HP-508. HP-508 is a representative IRS-2 activator (Housey and Balash, 2018; Housey and Balash, 2023). As described in this paper, HP-508 is classified as a vitality target enhancer (VTE).

[0082] Individuals with type 1 diabetes (T1D) wish to expand their remaining beta cell population in vitro for reimplantation into genetically identical tissue containing a sufficient number of beta cells to resolve their diabetes and normalize their blood sugar levels. This individual will act as both donor and recipient, receiving genetically identical beta cells upon completion of the procedure.

[0083] It has been shown that most patients with type 1 diabetes (T1D) have residual functional beta cells in their pancreas. The presence of these functional beta cells can be confirmed using a simple blood test that detects C-peptide, a byproduct of beta cell production of endogenous insulin. To determine whether residual beta cells are present in a patient's pancreas, a blood sample is drawn and the C-peptide level is determined. If a patient's C-peptide level is at or above 5% of the normal range (approximately 0.04 ng / ml or higher), the patient has functional beta cells and is eligible for surgery.

[0084] Next, a surgical biopsy sample is obtained from the pancreas. Typically, this involves the removal of the pancreatic tail, yielding approximately 20% to 25% of the total tissue. This is compatible with maintaining normal exocrine pancreatic function in patients with 75% to 80% remaining pancreatic tissue. Depending on the patient's C-peptide production levels, a smaller biopsy sample may be sufficient.

[0085] Figures 2 to 6 The results of obtaining human islets of Langerhans by surgically removing the pancreas from a cadaveric donor are shown. After resection, the pancreatic tissue was subjected to a standard islet tissue separation procedure identical to previously published procedures for allogeneic donor islet transplantation and autologous transplantation for chronic pancreatitis (McEachron and Bellin, 2018).

[0086] After islet isolation, the total number of isolated islets was determined using standard methods (Chetboun et al., 2023 and cited references). The islet tissue was then amplified under the culture conditions described below.

[0087] First, islets isolated from the biopsy sample were counted, and the initial total number of islets was recorded. The tissue was then placed in tissue culture medium supplemented with the IRS-2 activator HP-508. The tissue was then allowed to acclimatize to in vitro culture conditions for a period ranging from one hour to several days. The presence of HP-508 (VTE for these purposes) helps maintain the viability of the islet tissue, while routine passage culture under specific conditions allows for the selective removal of fibroblast-like cells mixed with the biopsy sample and dividing at a rate much higher than that of human islet tissue. The cell viability-enhancing ability of HP-508 is demonstrated in… Figure 2 middle.

[0088] Islet tissue maintained in the presence of IRS activators remained viable for more than a year and exhibited a slow but sustained growth pattern (Figure 3). In our laboratory, human islet tissue has been maintained under these conditions with sustained slow growth for more than three years; however, without the addition of cell growth enhancers as described herein, the growth rate was insufficient to support reimplantation surgery over a 12-month timeframe.

[0089] After culture adaptation was complete, a second substance enhancing the cell growth rate of islet tissue was added to the culture medium. As shown in Figure 3, the total surface area of ​​the tissue increased approximately fourfold over a 64-day period after the addition of the substance enhancing the cell growth rate of islet tissue. (We found that the determination of tissue surface area provides a usable estimate of the total wet tissue mass). This corresponds to a tissue doubling time of approximately 32 days. While still a slow division rate compared to rapidly dividing cell lines adapted to cell culture, this growth rate is sufficient to produce a total islet tissue mass equivalent to at least 400,000 islets over a six- to seven-month period following biopsy, starting from an initial islet mass of only 5,000 islet equivalents. Typical allogeneic transplantation typically uses 5,000 to 10,000 islet equivalents / kg body weight, which translates to approximately 350,000 to 700,000 islet equivalents / 70kg individual (McEachron and Bellin, 2018). If the initial biopsy produces a larger number of islets at the start, the minimum growth period will be shortened.

[0090] Substances that enhance cell growth rate in the presence of IRS activators include those that activate proteins that regulate cell cycle division (such as cell cycle-dependent kinases), as well as substances that inhibit proteins that normally act as inhibitors of cell cycle division. The latter include proteins that control metabolic states and proteins that prevent tumor formation (tumor suppressor factors).

[0091] like Figure 5As shown, the combination of IRS activators with substances that enhance cell growth leads to the formation of significantly sized islet tissue masses, which may even develop their own vascular-like structures as they grow and enlarge.

[0092] This disclosure enables long-term use of immunosuppressive drugs, thereby avoiding the numerous side effects associated with their use as described above. Furthermore, unlike iPSC-mediated methods, the selected cells do not need to dedifferentiate into iPSCs and then into insulin-producing "β-like" cells. This avoids the generation of mixed cell types whose long-term stability after implantation back into the patient is uncertain.

[0093] Example 2

[0094] Regeneration of dopaminergic neurons isolated from human substantia nigra

[0095] Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (a region deep in the human brain, directly above the basal ganglia). The activity of dopaminergic neurons in the substantia nigra has been shown to depend on IRS-2 (Xie et al., 2021; Ramalingam et al., 2017). Loss of IRS2 function leads to a reduction in dopaminergic neurons.

[0096] The compound HP-508 has been shown to activate IRS2. This compound acts as a VTE. Neurons were incubated in a medium containing the amount of HP-508 required to maintain healthy cells.

[0097] Patients with Parkinson's disease (PD) have lost dopaminergic neurons in the substantia nigra. A subset of healthy dopaminergic neurons is extracted from the patient via neurosurgical biopsy and expanded ex vivo. During the biopsy, an indwelling catheter is placed in the substantia nigra and maintained during the neuronal growth and expansion period. The obtained cells are expanded ex vivo, and subsequently, the expanded cells are reimplanted. Reimplantation of functional dopaminergic neurons helps improve previous symptoms. In this case, as in Example 1, the individual is both a donor and recipient of dopaminergic cells. Therefore, these cells are syngeneically identical to those in the surrounding tissue or autologous (genetically identical), thus reducing the likelihood of an autoimmune response in the subject.

[0098] After acclimatization in culture, a second substance that enhances neuronal cell growth is added to the culture medium. The appropriate number of neurons required for reimplantation is determined by the physician based on the degree of substantia nigra tissue loss observed in imaging studies. Furthermore, brain-derived neurotrophic factor (BDNF) has been shown to be reduced in patients with PD. Therefore, administration of a therapeutically effective amount of ex vivo expanded dopaminergic substantia nigra neurons will restore BDNF blood levels to normal (Palasz et al., 2020). Blood samples can be used for enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against identified BDNF antigens or fragments to determine when a sufficient number of cells should be reimplanted. Additionally, an indwelling catheter can be maintained for a sufficient period to allow for the reimplantation of additional cells until biomarkers associated with normal function are reached.

[0099] Example 3

[0100] Treatment of sensorineural hearing loss by in vitro amplification of human cochlear hair cells

[0101] Cells located in the cochlea of ​​the inner ear are essential for normal hearing. These cells contain hair follicles and are key cells that convert sound and vibrations into electrical signals within spiral ganglion neurons, which in turn transmit these signals to the brain. Sensorineural hearing loss is a major form of disease worldwide, and hearing aids remain the primary treatment. Currently, it is not possible to expand fully differentiated inner ear hair cells in vitro, and stem cell methods are being tested but have the same concerns as described in this article (Qi et al., 2023).

[0102] Using the method disclosed in this invention, a biopsy sample of inner ear hair cells is obtained by a surgical otolaryngologist, and in vitro amplification is performed as described in Examples 1 and 2 above. After in vitro amplification, a therapeutically effective amount of cells required to replenish the lost inner ear hair cells is re-infused into the cochlea in sufficient quantity to refill the inner wall of the cochlea with functional hair cells. Hearing can be restored in this way without the aid of external hearing aids. Summary of the Invention

[0104] Skilled researchers will readily recognize that this approach can be applied to other tissue reimplantation applications, such as multiple sclerosis with non-regenerative oligodendrocyte and Schwann cell demyelination, loss of specific motor neurons in spinal cord injury, and other diseases with progressive loss of specific functional cell types.

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[0150] All patents, patent applications, and publications cited herein, as well as the complete disclosures of electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in SwissProt, PIR, PRF, and PDB, and translations of annotated coding regions from GenBank and RefSeq), are incorporated herein by reference in their entirety. Supplementary materials cited in publications (such as supplementary tables, figures, materials and methods, and / or experimental data) are also incorporated herein by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall prevail. The foregoing detailed descriptions and examples are given only for clarity of understanding. Unnecessary limitations should not be construed therein. This disclosure is not limited to the exact details shown and described, and variations that will be apparent to those skilled in the art will be included within the scope of this disclosure as defined by the claims.

[0151] Unless otherwise indicated, all figures representing quantities, molecular weights, etc., of components as used in this specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary according to the desired characteristics to be obtained through this disclosure. At least, and without attempting to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0152] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​illustrated in specific examples are reported as precisely as possible. However, all values ​​inherently contain ranges that are necessarily caused by the standard deviations found in their respective test measurements.

[0153] All headings are for the reader's convenience and, unless otherwise specified, should not be used to limit the meaning of the text following the heading.

Claims

1. A method of expanding tissue or cells ex vivo, the method comprising: (a) providing a biopsy sample of the tissue from an individual having a disease, wherein the disease has resulted in loss of the tissue; (b) expanding the tissue or cells ex vivo to obtain expanded tissue or cells, wherein the expanding comprises culturing the tissue in the presence of a compound that activates insulin receptor substrate (IRS) proteins and a substance that enhances cell division; and (c) reimplanting a therapeutically effective amount of the expanded tissue or cells into the individual.

2. The method of claim 1, wherein the disease comprises type 1 diabetes or Parkinson’s disease.

3. The method of claim 1, wherein the substance comprises siRNA that reduces expression of TP53, STK4, VGLL4, or TSC-1.

4. The method of claim 1, wherein the therapeutically effective amount of the expanded tissue comprises a mass equivalent to at least 400,000 cells.

5. The method of claim 1, wherein the individual is a human.

6. A method of treating a subject having diabetes, the method comprising: (a) providing a biopsy sample of pancreatic tissue from an individual having diabetes; (b) isolating islets contained in the biopsy sample; (c) expanding the islets ex vivo to obtain expanded islets, wherein the expanding comprises culturing the islets in the presence of a compound that activates insulin receptor substrate (IRS) proteins and a substance that enhances cell division; and (d) reimplanting a therapeutically effective amount of the expanded islets into the individual.

7. The method of claim 6, wherein the substance comprises siRNA that reduces expression of TP53, STK4, VGLL4, or TSC-1.

8. The method of claim 6, wherein the therapeutically effective amount of the expanded islets comprises a mass equivalent to at least 400,000 islets.

9. The method of claim 6, wherein the reimplanting comprises portal vein infusion, omental injection, or a combination thereof.

10. The method of claim 6, wherein the method further comprises determining whether blood glucose control of the subject is restored.

11. The method of claim 6, wherein the subject is a human.

12. A method of expanding human insulin-producing tissue ex vivo, the method comprising: (a) providing a biopsy sample of pancreatic tissue from a subject having diabetes who has a relative or absolute deficiency of insulin; (b) isolating islets contained in the biopsy sample; (c) expanding the islets to obtain expanded islets, wherein the expanding comprises culturing the tissue in the presence of a compound that activates insulin receptor substrate (IRS) proteins and a substance that enhances cell division; and (d) confirming that the expanded islets continue to produce insulin.

13. The method of claim 12, wherein the substance comprises siRNA that reduces expression of TP53, STK4, VGLL4, or TSC-1.

14. The method of claim 12, wherein the therapeutically effective amount of the expanded pancreatic islets comprises a mass equivalent to at least 400,000 islets.

15. The method of claim 12, wherein the method further comprises re-implanting the expanded pancreatic islets.

16. The method of claim 15, wherein the re-implanting comprises portal vein infusion, omental injection, or a combination thereof.

17. The method of claim 12, wherein the biopsy sample is obtained from a human.

18. A method of treating a subject having Parkinson’s disease, the method comprising: (a) providing a biopsy sample of a substantia nigra region of the brain of the subject, wherein the biopsy sample comprises dopaminergic neurons; (b) expanding substantia nigra tissue ex vivo to yield expanded substantia nigra tissue, wherein the expanding comprises culturing the tissue in the presence of a compound that activates insulin receptor substrate (IRS) proteins and a substance that enhances cell division; and (c) re-implanting a therapeutically effective amount of the expanded tissue into the individual.

19. The method of claim 18, wherein the substance comprises an siRNA that reduces expression of TP53, STK4, VGLL4, or TSC-1.

20. The method of claim 18, wherein the therapeutically effective amount of the expanded tissue comprises a mass equivalent to at least 400,000 cells.

21. The method of claim 18, wherein the re-implanting comprises transcranial infusion, intracranial injection, or a combination thereof.

22. The method of claim 18, further comprising determining whether substantia nigra function is restored in the subject.

23. The method of claim 18, wherein the biopsy sample is obtained from a human.

24. A method of expanding human dopaminergic substantia nigra neuron tissue ex vivo, the method comprising: (a) providing a biopsy sample of substantia nigra comprising dopaminergic neurons from a subject having Parkinson’s disease; (b) expanding the biopsy sample of the substantia nigra ex vivo to yield expanded substantia nigra cells, wherein the expanding comprises culturing the biopsy sample in the presence of a compound that activates insulin receptor substrate (IRS) proteins and a substance that enhances cell division; and (c) confirming that the expanded substantia nigra tissue continues to produce dopamine.

25. The method of claim 24, wherein the substance comprises a mass equivalent to at least 400,000 cells.

26. The method of claim 24, wherein the biopsy sample is obtained from a human.

27. The method of claim 24, further comprising re-implanting a therapeutically effective amount of the expanded cells into the individual.

28. The method of claim 27, wherein the re-implanting comprises transcranial infusion, intracranial injection, or a combination thereof. ​

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