Selective functional enhancement of stem cells and or their genetic material, with gene transfection for short-and long-term treatment of age-related disease states

Abstract

The function of enhancing stem cells with a specific transfected human gene type into a stem cell of choice to allow for the regenerative potential of the selected stem cell to be greatly enhanced by combining both gene and stem cell therapies together. Specific genes for selected protein production are cultured with a stem cell of choice which then produces a supernatant composed of an exosome-gene complex. This exosome-gene complex can also be used as a therapeutic gene therapy on its own or in combination with a stem cell gene complex together for a specific cellular effect, organ or tissue of regenerative effect or general systemic result.

Description

[0001] File No. 0425.141 (PCT)

[0002] SELECTIVE FUNCTIONAL ENHANCEMENT OF STEM CELLS AND OR THEIR GENETIC MATERIAL, WITH GENE TRANSFECTION FOR SHORT- AND LONG-TERM TREATMENT OF AGE-RELATED DISEASE STATES.

[0003] BACKGROUND OF THE INVENTION

[0004] Stem cells have shown great promise in regenerative medicine for multiple different disease processes caused by the general effects of the cellular aging process. At the same time genetic therapies alone have also been shown to be significantly important in treatments used for specific inherited disease states as well as cancer.

[0005] The use of exosome therapy, the product of stem cells has also been shown to have regenerative effects on both cell function and certain diseases related to the aging process.

[0006] This invention describes the concept of enhancing stem cell function with a specific manufactured human gene type which will allow for the regenerative potential of stem cells to be greatly enhanced by combining both gene and stem cell therapies together. The combination of these two nascent technologies creates a synergistic effect for regenerative cellular therapy far beyond their effectiveness alone.

[0007] The use of exosome-derived macrovesicles has been successfully used by the present inventors Vincent Giampapa and John Greco in U.S. Patent No. 10,772,911 (2020). Also, the Use of Enhancing Telomere Cell has been used by Vincent Giampapa and Linda Crouse of U.S. Patent No. 10,098,922 (2018).

[0008] In other pertinent part is U.S. 2014 / 0010801 A1 of Niedernhofer, et al regarding Composition and Methods or Restoring or Rejuvenating Stem Cells. And is U.S. 2022 / 0251603 A1 to Parrish, et al regarding System and Methods for Gene Therapy via Genetically Modified Viral Vectors. And yet further U.S. 2022 / 0325258 A1 of Kogut, et al regarding Methods for Cell and Tissue Rejuvenation; and U.S. 2022 / 0136011 A1 of Kalluri for Telomerase-Containing Exosomes for Treatment of Age-Related Organ Dysfunction. Also, China Patent No. 112695049 A of Liu Minglu, et al., for Fusion Gene and Plasmid for Mesenchymal

[0009] Stem Cells.

[0010] SUMMARY OF THE INVENTION

[0011] The invention also describes the use of specific human genes that can be used with exosomes alone forming an exosome-gene complex, or with the combination of stem cell- gene enhancement in combination with exosome-gene complexes.

[0012] The focus of this invention is to help restore enhanced cellular function and the physical functional loss occurring in humans due to the general aging process with genetically enhanced stem cells, with us without exosome-gene complexes.

[0013] The functional losses in humans over time which are of main concern here include, sarcopenia and frailty, cognitive impairment, central brain regulatory mechanisms, dementia, immunosenescence, as well as the loss of cellular and general body energy production. All these effects of aging decrease quality of life and health span as well as longevity. They have been well documented in many recent peer reviewed science publications. The cellular and physical loss of functions cause trillions of dollars in global healthcare costs in the aging population. This technology may be a large benefit in decreasing the long-term care of the global aging population.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1- Is a schematic view of a cell, nucleus, chromosome and DNA structure.

[0016] Fig. 2 - Is a schematic view of the use of a viral DNA and vector binds to cell membrane and the process thereafter set forth below.

[0017] Fig. 3 - Is a schematic view of the aging clocks of the human body.

[0018] Fig. 4 - is a cell-based direct delivery system

[0019] Fig. 5 - is a schematic view of a basic structure of a MSC cell including exosomes depicting the surface and core proteins along with key immune and growth factors.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Gene vectors 32 (see Fig. 2) can be a viral or plasmid 18A based, as well as other means of transfection 34 mentioned here. The gene 16 transferred into the nucleus 12 does not incorporate into the cellular genetics 10 in any significant levels but remains in the nucleus 12 as an episome 39 or extra piece of DNA 18 (see Figs. 2 and 6) which will then produce the protein of choice. The protein may leave cell 10 and makes its way into the systemic vascular system of the person (see Fig. 4), or animal, deploying its effects throughout the body. It may also remain in the cell to alter specific functions like increased NAD and or ATP production or even induce the production of more cellular organelles like mitochondria

[0022] Specific genes for selected protein production may be cultured with a stem cell 11 of choice (see Fig. 4 and 5) which will then produce a supernatant composed of an exosome-gene complex 16A / 39 (see Fig. 2). This exosome-gene complex may also be used as a therapeutic gene therapy on its own or in combination with the stem cell gene complex together for a specific cellular effect, organ or tissue regenerative effect or general systemic effects.

[0023] The effects of the central aging clock (see Fig. 3) located in the hypothalamus 48 and the memory center in the hippocampus 50, control multiple aspects of the body’s natural rhythms related to health and aging. Loss of neural stem cells over time with the aging process causes these centers to dysregulate and eventually become inefficient to maintain normal health. Restoration of the cells lining the hypothalamus 48, the neural stem cells 44, with gene enhanced stem cells 34 like mesenchymal stem cells 38 (see Fig. 5) and there exosomes 39 loaded with the klotho gene 41 (see Fig. 1), can rejuvenate the function of the hypothalamus 48 to reset the central aging clock 49, as well as the hippocampus 50 to enhance memory and cognition 54 normally lost with the aging process. See Fig. 3

[0024] Described in this patent is a gene therapy technique to treat or prevent a disease or physiological function loss, caused by the human aging process. This is accomplished by inserting a specific gene into a patients stem cell of choice. Stem cells can be genetically modified to carry therapeutic genes 34. These modified or enhanced stem cells can then be used to regenerate damaged tissues, organs or even restore cellular function (see Fig. 3). The techniques described herein can be accomplished via:

[0025] 1 - an AAV - (attenuated adenovirus) or CMV- (cytomegalovirus) vector delivery systems 36A for long -term functional enhancement (see Fig. 4) or,

[0026] 2- a plasmid delivery system, for a short-term delivery system (see Fig. 2).

[0027] Both vector types can use selected genes of choice delivered into stem cells of different cell lineage. In this case we are describing human adult stem cells, both allogeneic and autologous stem cells including but not limited to hematopoietic stem cells, endothelial progenitor cells, and mesenchymal stem cells 38 (see Fig. 5). The source of these stem cells can be from peripheral blood, bone marrow or fat cells, or from umbilical cord or other humans tissue sources or induced pluripotent stem cells this can be accomplished for human sources most readily via apheresis collection after mobilization with any number of mobilizing agents including neurogenic to obtain a combination of multiple stem cell types 43 (see Fig. 4).

[0028] The goal of this process is to deliver or insert a gene of choice within the nuclear compartment of the stem cell, as an episome 39, virtually nonintegrating with the original nuclear DNA 18. This acts as an additional artificial chromosome 14. These transfected cells can then be used systemically or locally to enhance stem cell function or suppress a given protein or restore specific cellular function as in increased muscle mass 52 (see Fig. 3) or enhanced cognition 54 (see Fig. 3) as well as immune enhancement 13 (see Fig. 5)

[0029] The transfected cells 34 (see Fig. 4) can then be culture expanded to create more numbers of the enhanced cells type for specific purposes 40 / 55 and then reinfused intravenously and or injected into aged local tissues, injected into lymphatics or the spinal canal 53 as well as other anatomical sites (see Fig. 3). More specifically certain stem cell lines can be treated with gene transfection with specific genes for a specific purpose. For example, hematopoietic stem cells, which form all cell types involved in immune system 13, can be treated with hTERT to extend their telomere length 39 and allow them to make more copies of themselves for a much longer time than normally possible, therefore helping to restore immune function and avoid immunosenescence, which occurs to all human adults as they age. hTERT or telomerase 34 / 39 (see Figs 2 and 6) is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG 28, 26, 22 (see Fig. 1 ). The enzyme consists of a protein component with reverse transcriptase (hTERT) activity 16A / 34, encoded by this gene, and a RNA component 46 that serves as a template for the telomere repeat (see Fig. 5). Mesenchymal stem cells 38 (see also Fig. 5) can be treated with flotillin protein 47 and or PGC-1 alpha which can be injected either locally into muscle or given IV systemically to restore lost muscle mass and strength 52.

[0030] These same mesenchymal stem cells 38 may also be given to avoid sarcopenia and frailty that occurs in all adults over time. The same mesenchymal stem cells can also be transfected with alpha klotho 41 (see Fig. 1 ) and injected IV along with focused ultrasound which can help guide them to specific brain centers like the hypothalamus and hippocampus to restore the central aging clock 49 nucleus involved in multiple physical functions, (see Fig 3) to help restore the neural nuclei responsible for maintaining general homeostasis of the aging process including memory decline as well as dementia and Parkinson disease.

[0031] Endothelial progenitor cells and other precursors to blood vessel formation can be transfected with hTERT also to enhance blood vessel 49 (transferrin) repair (see Fig. 5) and angiogenesis which is lost over time due to the aging process.

[0032] Selective immune cells like natural killer cells can also be transfected with hTERT to function as a senolytic therapy to remove senescent cells, remove viral infections, as well as cancer cells and bacterial infections.

[0033] The concepts presented here are not limited to the genes mentioned in this application but may apply to multiple other genes that can be used for different organ regeneration like the liver 55, thymus, pancreas and other organs (see Fig. 3) and tissues not mentioned here. In general, the general process described here can be used to enhance stem cell function not mentioned in this preliminary application.

[0034] The general concepts revealed here can also be applied not just to humans but to other non-human primates and animals in general including dogs, cats, and horses.

[0035] The intended use of this therapy is to create enhanced stem cell function on a long-term or permanent basis using different gene- based transfection technologies and different plasmid-based gene transfection technologies. Both technologies may incorporate a start or stop gene or other technologies to control the magnitude of gene expression desired for a given purpose or amount of time if needed for a given condition or effect.

[0036] The use of exosome products 39 from the transfected stem cell 36 / 43 can also be used for therapeutic effect with or without the transfected stem cells.

[0037] Although the present invention has been illustrated and described herein with reference to preferred embodiments and specific examples thereof, it will be readily apparent to those of ordinary skill in the art that other embodiments may perform similar functions and like results. All such equivalent embodiments and examples are within the spirit and scope of the present invention are contemplated thereby and are intended to be covered by the following claims

Claims

CLAIMS1 . An adult stem cell therapy, comprising;(a) using a mesenchymal stem cell (MSC) as a subset of adult stem cells (ASC);(b) using exosomal vesicles (EeV) extracting a basic structure of MSC cells;(c) providing a telomere analysis of a degree of elongation of a telomere; and(d) using a vector mediator to provide a restoration of telomerase reverse transcriptase (hTERT) or enzyme thereof using a telomerase RNA component (TERC).

2. The therapy as recited in claim 1 , comprising:(a) having a vector subunit of an enzyme of a telomerase cell membrane including a vector;a EeV of a nucleus thereof and a cell of a membrane including an episome of a DNA of a cell using a protein of choice;(b) providing the EeV as a degenerating a vector package having said vector thereof; and(c) combining said protein into a new gene of the cell.

3. The therapy as recited in claim 2, comprising: extracting said EeV and its DNA into MSG cells.

4. The therapy as recited in claim 3, comprising: using a klotho gene within said MSC cells having said exosomes or EcV.

5. The therapy is recited in claim 4, comprising: using a vector delivery of a plasmid of the cell.

6. The therapy of claim 4, comprising: said klotho gene transfection in said MSC cells.

7. The therapy as recited in claim 6, comprising: using AAV or CMV into said vector or vector package.

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