METHODS AND MATERIALS FOR REDUCING AGE-RELATED STRIATED MUSCLE AND COGNITIVE DECLINE
20220008520 · 2022-01-13
Inventors
- Fabrisia Ambrosio (Pittsburgh, PA, US)
- Aaron Barchowsky (Pittsburgh, PA, US)
- Amin Cheikhi (Pittsburgh, PA, US)
- Rada Koldamova (Bethel Park, PA, US)
- Iliya Lefterov (Bethel Park, PA, US)
- Amrita Sahu (Pittsburgh, PA, US)
Cpc classification
A01K2217/077
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
A61K38/47
HUMAN NECESSITIES
C12N9/2402
CHEMISTRY; METALLURGY
A61K9/127
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
International classification
A61K38/47
HUMAN NECESSITIES
A61K9/127
HUMAN NECESSITIES
Abstract
This document provides methods and materials for treating aging. For example, a mammal having, or at risk for developing, an age-related impairment (e.g., age-related cognitive decline) can be treated by increasing the level of one or more myokine polypeptides (e.g., one or more Klotho polypeptides) within cells within the mammal. This document also provides methods and materials for increasing the ability of muscle progenitor cells to regenerate muscle cells by increasing the level(s) of one or more myokine polypeptides (e.g., an α-Klotho polypeptide) within a muscle progenitor cell.
Claims
1. A method for reducing sarcopenia or age-related cognitive decline within a mammal, wherein said method comprises administering exosomes comprising an α-Klotho polypeptide or a nucleic acid encoding said α-Klotho polypeptide to said mammal.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said method comprises administering exosomes comprising said α-Klotho polypeptide to said mammal.
4. The method of claim 1, wherein said method comprises administering exosomes comprising said nucleic acid to said mammal.
5-9. (canceled)
10. A method for increasing the ability of muscle progenitor cells to regenerate muscle cells within a mammal having a muscle impairment, wherein said method comprises: (a) identifying said mammal as having said muscle impairment, and (b) administering an α-Klotho polypeptide to said mammal.
11. (canceled)
12. The method of claim 10, wherein said mammal is a human.
13. The method of claim 10, wherein said muscle impairment is sarcopenia.
14-20. (canceled)
21. A method for increasing the ability of muscle progenitor cells to regenerate muscle cells within a mammal, wherein said method comprises administering an exosome comprising an α-Klotho polypeptide or a nucleic acid encoding said α-Klotho polypeptide to said mammal.
22. The method of claim 21, wherein said mammal is a human.
23. The method of claim 21, wherein said mammal has sarcopenia.
24. The method of claim 21, wherein said method comprises administering an exosome comprising said α-Klotho polypeptide to said mammal.
25. The method of claim 21, wherein said method comprises administering an exosome comprising said nucleic acid to said mammal.
26-49. (canceled)
Description
DESCRIPTION OF THE DRAWINGS
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[0027] (J) RT-PCR analysis of mtDNA damage in MPCs isolated from wild-type and Kl+/− mice. (*p<0.05, **p<0.01, ****p<0.0001).
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DETAILED DESCRIPTION
[0058] This document provides methods and materials for treating aging (e.g., for treating aged mammals). For example, a mammal having, or at risk for developing, an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) can be treated by increasing the level of one or more myokine polypeptides (e.g., one or more Klotho polypeptides) in one or more cells within the mammal. In some cases, one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) to treat the mammal. In some cases, nucleic acid encoding one or more myokine polypeptides (e.g., nucleic acid encoding one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) to treat the mammal. In some cases, an exosome containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid (e.g., mRNA) encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) to treat the mammal. In some cases, a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) to treat the mammal.
[0059] This document also provides methods and materials for increasing the ability of stem cells (e.g., MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a mammal (e.g., a human) having an impairment or injury (e.g., a muscle impairment). For example, one or more myokine polypeptides (e.g., one or more Klotho polypeptides such as an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., nucleic acid encoding one or more Klotho polypeptides such as an α-Klotho polypeptide), exosomes containing one or more myokine polypeptides and/or nucleic acid encoding one or more myokine polypeptides, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be used to increase the ability of stem cells (e.g., MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a mammal (e.g., a human) having an impairment or injury (e.g., a muscle impairment) to treat that an impairment or injury. Examples of muscle impairments that can be treated as described herein include, without limitation, sarcopenia, traumatic muscle injury, myopathies, and post-surgical muscle injury.
[0060] In general, the ability of stem cells (e.g., MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a mammal (e.g., a mammal having a muscle impairment) can be increased by increasing the level of one or more myokine polypeptides (e.g., a Klotho polypeptide such as an α-Klotho polypeptide) within a muscle progenitor cell. The level of one or more myokine polypeptides (e.g., a Klotho polypeptide such as an α-Klotho polypeptide) within a stem cell (e.g., an MPC) can be increased using any of the methods or materials described herein. For example, the methods or materials described herein for increasing the level of one or more myokine polypeptides (e.g., a Klotho polypeptide such as an α-Klotho polypeptide) to treat an age-related impairment (e.g., sarcopenia and/or age-related cognitive decline) can be applied to stem cells (e.g., MPCs) to increase the ability of stem cells (e.g., MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a mammal. The level of one or more myokine polypeptides (e.g., a Klotho polypeptide such as an α-Klotho polypeptide) within a stem cell (e.g., an MPC) can be increased by administering one or more myokine polypeptides (e.g., one or more Klotho polypeptides) to a mammal having stem cells, by administering nucleic acid (e.g., mRNA) encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) to a mammal having stem cells, and/or by administering an exosome containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid (e.g., mRNA) encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) to a mammal having stem cells. The administration of exosomes containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid (e.g., mRNA) encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can result in the exosomes crossing the blood brain barrier to deliver the contents to cells (e.g., neurons, microglia, and astrocytes) within the brain of a mammal (e.g., a human).
[0061] Examples of stem cells that can be treated as described herein to increase regenerative capabilities include, without limitation, epithelial stem cells, MPCs, neuronal stem cells, and hematopoietic stem cells. In some cases, increasing the ability of epithelial stem cells to regenerate epithelial cells can be used to improve wound healing. In some cases, the methods and materials described herein can be used to increase the ability of aged stem cells (e.g., aged MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a mammal (e.g., a human) having an impairment or injury (e.g., a muscle impairment). For example, the methods and materials described herein can be used to increase the ability of aged stem cells (e.g., aged MPCs) to regenerate more differentiated cells (e.g., muscle cells) within a human that is over the age of 20, 30, 40, 50, 60, or 70 years and has an impairment or injury (e.g., a muscle impairment).
[0062] The term “increased level” as used herein with respect to a level of a myokine polypeptide (e.g., a Klotho polypeptide) refers to any level that is greater than the median level of that myokine polypeptide as typically observed in a mammal that does not have an age-related impairment. Control samples can include, without limitation, samples from young mammals. It will be appreciated that levels from comparable samples or tissues are used when determining whether or not a particular level is an increased level.
[0063] Any appropriate mammal having, or at risk for developing, an age-related impairment can be treated as described herein. Examples of mammals having, or at risk for developing, an age-related impairment that can be treated as described herein (e.g., by increasing the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within the mammal) include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having, or at risk for developing, an age-related impairment can be treated by increasing the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within that human.
[0064] When treating a mammal (e.g., a human) having, or at risk for developing, an age-related impairment as described herein (e.g., by increasing the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within the mammal), the age-related impairment can be any type of age-related impairment. In some cases, an age-related impairment can be associated with reduced or eliminated levels of one or more myokine polypeptides. For example, an age-related impairment can be associated with methylation of a promoter that directs expression of one or more myokine polypeptides such that the methylated promoter results in reduced or eliminated levels of one or more myokine polypeptides. In some cases, an age-related impairment can be a degenerative disease or condition. Examples of age-related impairments include, without limitation, declines in cellular regeneration, cognitive declines, atrophy, wound healing, arteriosclerosis, osteoporosis, muscle impairments associated with aging (e.g., sarcopenia), and impaired regenerative responses. An age-related impairment can affect any part of a mammal (e.g., any part of a mammal's body). Examples of parts of a mammal that can be affected by an age-related impairment include, without limitation, muscles (e.g., skeletal muscles, smooth muscles, and cardiac muscles), blood vessels (e.g., arteries), nerves, bones, or skin. In some cases, an age-related impairment can be an age-related decline in muscle regeneration (e.g., impaired muscle regeneration). In some cases, an age-related impairment can be an age-related cognitive decline (e.g., impaired cognitive function).
[0065] In some cases, methods described herein can include identifying a mammal (e.g., a human) as having, or as being at risk for developing, an age-related impairment. Any appropriate method can be used to identify a mammal as having an age-related impairment. For example, a reduced level of one or more myokine polypeptides in a sample obtained from a mammal can be used to identify a mammal having an age-related impairment. For example, the presence of methylation on a promoter that directs expression of a myokine polypeptide (e.g., a methylated myokine promoter such as a methylated Klotho promoter) in a sample obtained from a mammal can be used to identify a mammal having an age-related impairment. A sample can be any appropriate sample. In some cases, a sample can be a fluid sample (e.g., a blood sample). In some cases, a sample can be tissue sample (e.g., a biopsy). Examples of samples that can be obtained from a mammal and assessed for a reduced level of one or more myokine polypeptides and/or the presence of methylation on myokine promoter include, without limitation, blood samples (e.g., whole blood, serum, and plasma), muscle tissue samples, and urine samples.
[0066] Once identified as having, or as being at risk for developing, an age-related impairment, a mammal (e.g., a human) can be administered, or instructed to self-administer, one or more myokine polypeptides (e.g., one or more Klotho polypeptides). For example, one or more myokine polypeptides can be administered to a mammal in need thereof (e.g., a mammal having, or at risk for developing, an age-related impairment). For example, a human having, or at risk for developing, an age-related impairment can be treated by administering one or more myokine polypeptides, nucleic acid encoding one or more myokine polypeptides, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of one or more myokine polypeptides to increase the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within that human.
[0067] In some cases, one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment to treat the mammal. For example, a mammal having, or at risk for developing, an age-related impairment can be administered or can self-administer a composition containing one or more myokine polypeptides. In some cases, a composition containing one or more myokine polypeptides (e.g., a composition containing a Klotho polypeptide) can be administered to a mammal having an age-related impairment to increase the level of one or more myokine polypeptides (e.g., a Klotho polypeptide) within that human.
[0068] A myokine polypeptide can be any appropriate myokine polypeptide. In some cases, a myokine polypeptide can be an anti-aging myokine. In some cases, a myokine polypeptide can be an exercise-induced myokine polypeptide (e.g., a myokine polypeptide whose cellular expression of the myokine polypeptide is driven by physical exertion and/or skeletal muscle contraction). In some cases, a myokine polypeptide can be a circulating myokine polypeptide (e.g., a myokine polypeptide present in the bloodstream of a mammal). In some cases, a myokine polypeptide can be from about 5 kDa to about 140 kDa (e.g., from about 5 kDa to about 135 kDa, from about 15 kDa to about 140 kDa, from about 50 kDa to about 140 kDa, or from about 120 kDa to about 135 kDa). In some cases, a myokine polypeptide can have autocrine, paracrine and/or endocrine effects. Examples of myokine polypeptides that can be used as described herein (e.g., to treat a mammal having, or at risk for developing, an age-related impairment) include, without limitation, interleukins (ILs; e.g., IL-6), Klotho (e.g., α-Klotho, β-Klotho, and γ-Klotho), GDF-11, and brain-derived neurotrophic factor (BDNF). For example, a mammal having, or at risk for developing, an age-related impairment can be administered or can self-administer one or more Klotho polypeptides (e.g., one or more α-Klotho polypeptides). An example of a Klotho polypeptide that can be used as described herein includes, without limitation, a human Klotho polypeptide having the amino acid sequence set forth in National Center for Biotechnology Information (NCBI) GenBank® Accession No. NP_004786.2. A representative human Klotho polypeptide sequence is set forth in
[0069] In some cases, once identified as having, or as being at risk for developing, an age-related impairment, a mammal (e.g., a human) can be administered, or instructed to self-administer, nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides). For example, nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment to treat the mammal. In some cases, nucleic acid encoding one or more myokine polypeptides can be administered to a mammal having, or at risk for developing, an age-related impairment to increase the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within that human.
[0070] A nucleic acid encoding a myokine polypeptide (e.g., a Klotho polypeptide) can be any appropriate nucleic acid. A nucleic acid encoding a myokine polypeptide can encode any myokine polypeptide described herein. In some cases, a nucleic acid encoding a myokine polypeptide can encode a Klotho polypeptide (e.g., an α-Klotho polypeptide). An example of a nucleic acid encoding a Klotho polypeptide includes, without limitation, nucleic acid encoding a human Klotho sequence as set forth in GenBank® Accession No. NM_004795.3. A representative nucleic acid sequence encoding a human Klotho polypeptide is set forth in
[0071] In some cases, a nucleic acid encoding a myokine polypeptide (e.g., a Klotho polypeptide) can be in a nucleic acid vector (e.g., an expression vector). In some cases, a vector can be a plasmid. In some cases, a vector can be viral vector (e.g., a lentiviral vector). Examples of viral vectors that can be used to deliver nucleic acid encoding one or more myokine polypeptides (e.g., a Klotho polypeptide) to a mammal to treat an age-related impairment as described herein include, without limitation, adenoviral vectors, adeno-associated viral vectors (e.g., AAV8 viral vectors and chimeric AAV2/AAV8 viral vectors), lentiviral vectors, herpes viral vectors, retroviral vectors, and vaccinia viral vectors.
[0072] An expression vector (e.g., viral vector) can include one or more elements necessary for expressing a polypeptide (e.g., a myokine polypeptide) from a nucleic acid sequence within the vector (e.g., a ribosomal binding site and start codon, a termination codon, and a transcription termination sequence). In cases where a nucleic acid encoding a myokine polypeptide is a vector, the vector also can include one or more regulatory elements (e.g., enhancers and promotes) that can enhance expression of a polypeptide (e.g., a myokine polypeptide) from a nucleic acid sequence within the vector. A promoter can be a constitutive promoter or an inducible promoter. A promoter can be a ubiquitous promoter or a tissue/cell-specific promoter (e.g., a muscle-specific promoter). An example of a promoter that can increase expression of a polypeptide (e.g., a myokine polypeptide) from a nucleic acid sequence within a vector includes, without limitation, a Pitx3 muscle-specific promoter.
[0073] In cases where a nucleic acid encoding a myokine polypeptide is a vector, the vector also can include an origin of replication, a selectable marker, and/or a nucleic acid encoding a detectable label.
[0074] In some cases, once identified as having, or as being at risk for developing, an age-related impairment, a mammal (e.g., a human) can be administered, or instructed to self-administer, exosomes containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides). For example, exosomes containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal in need thereof (e.g., a mammal having, or at risk for developing, an age-related impairment). For example, a human having, or at risk for developing, an age-related impairment can be treated by administering exosomes containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides). In some cases, an exosome can contain mRNA encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides).
[0075] Any appropriate method can be used to obtain exosomes containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides). For example, cells (e.g., muscle cells) in cultured in a manner to accumulate exosomes in culture. In some cases, the cultured cells can be genetically manipulated to express one or more myokine polypeptides (e.g., one or more Klotho polypeptides) from exogenously added nucleic acid. Once produced, the exosomes can be isolated from the cell culture supernatants. Any appropriate method can be used to obtain or enrich a sample for exosomes. For example, cell sorting techniques can be used to obtain particular exosomes.
[0076] In some cases, exosomes isolated from cells can be treated in a manner that loads particular contents into the exosome. For example, engineered skeletal muscle or muscle stem cell exosomes can be loaded with exogenous myokine polypeptides (e.g., an α-Klotho polypeptide) or nucleic acid encoding a myokine polypeptide (e.g., nucleic acid encoding an α-Klotho polypeptide). In some cases, exosomes can be designed to have one or more rabies virus glycoprotein (RVG) peptides attached to their surface to deliver the myokine cargo to neurons, microglia, and oligodendrocytes after administration (e.g., an intravenous injection). Other exosome surface modifications or attachments of molecules on exosomal surfaces can be used to fine tune the stability of the exosomes in vivo, the pharmacokinetics of the exosomes, and/or the biodistribution of the exosomes.
[0077] In some cases, synthetically generated vesicles can be used in place of exosomes. For example, a synthetically generated vesicle having dimensions similar to those of exosomes can be made to contain (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and used as described herein. Any appropriate method can be used to make synthetically generated vesicles containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides). For example, high payloads of (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more
[0078] Klotho polypeptides) can be stably accommodated by several synthetic liposomal formulations or exosomes-mimetics of semi-synthetic origin (e.g., using lipids extracted from specific tissues, cells, or extracellular vesicles) that overcome cellular/tissue uptake obstacles and promote better biodistribution. In some cases, synthetic liposomal formulations or exosomes-mimetics can provide efficient systemic delivery within a mammal's brain or other target distal organs in a manner that minimizes any collateral systemic side effects.
[0079] In some cases, an exosome (and/or synthetically generated vesicle) containing (a) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (b) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered systemically to deliver contents to cells within a mammal's brain or other distal organs.
[0080] In some cases, a gene editing system designed to alter or eliminate one or more methylation sites (e.g., a 10 to 50 base pair region containing multiple CpG sites, a 10 to 100 base pair region containing multiple CpG sites, a 10 to 150 base pair region containing multiple CpG sites, a 10 to 200 base pair region containing multiple CpG sites, a 25 to 50 base pair region containing multiple CpG sites, a 25 to 100 base pair region containing multiple CpG sites, a 25 to 150 base pair region containing multiple CpG sites, a 25 to 200 base pair region containing multiple CpG sites, a 50 to 100 base pair region containing multiple CpG sites, a 50 to 150 base pair region containing multiple CpG sites, a 50 to 200 base pair region containing multiple CpG sites, a 100 to 150 base pair region containing multiple CpG sites, or a 100 to 200 base pair region containing multiple CpG sites) of a promoter that directs expression of one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be administered to a mammal having, or at risk for developing, an age-related impairment to treat the mammal. For example, a mammal (e.g., a human) having, or at risk for developing, an age-related impairment can be administered or can self-administer a gene editing system designed to replace one or more methylation sites of a promoter that directs expression of one or more myokine polypeptides with a promoter sequence that lacks one or more methylation sites to increase the expression level of one or more myokine polypeptides (e.g., a Klotho polypeptide) in one or more cells within that mammal (e.g., human). In some cases, the region from about −1 (with respect to the ATG start site) to about −500 (e.g., from −1 to −500, from −1 to −450, from −1 to −400, from −1 to −350, from −1 to −300, from −1 to −250, from −1 to −200, from −1 to −150, from −1 to −100, from −1 to −50, from −10 to −500, from −10 to −450, from −10 to −400, from −10 to −350, from −10 to −300, from −10 to −250, from −10 to −200, from −10 to −150, from −10 to −100, from −10 to −50, from −50 to −500, from −50 to −450, from −50 to −400, from −50 to −350, from −50 to −300, from −50 to −250, from −50 to −200, from −50 to −150, or from −50 to −100) of a promoter that directs expression of one or more myokine polypeptides (e.g., one or more Klotho polypeptides) can be removed. Examples of methylation sites (e.g., CpG dinucleotides) within a promotor that can drive expression of a human Klotho polypeptide that can be removed or replaced as described herein to increase the expression of Klotho polypeptides include, without limitation, those methylation sites within the underlined sequence (SEQ ID NO:4) of the sequence set forth in
[0081] A gene editing system designed to remove or replace one or more methylation sites of a promoter that directs expression of one or more myokine polypeptides can be any appropriate gene editing system. Examples of gene editing systems that can be designed to reduce or eliminate methylation of a promoter that directs expression of one or more myokine polypeptides include, without limitation, zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 systems. When a CRISPR/Cas9 system is used to reduce or eliminate methylation of a promoter that directs expression of one or more myokine polypeptides, the Cas9 component of a CRISPR/Cas9 system can be any appropriate Cas9 (e.g., a Staphylococcus aureus Cas9 (saCas9)). The nucleic acid and/or polypeptide sequences of such genome editing molecules can be as described elsewhere (see, e.g., Mani et al., Biochemical and Biophysical Research Communications, 335:447-457 (2005); Campbell et al., Circulation Research, 113:571-587 (2013); Cong et al., Science, 339:819-823 (2013); and Ran et al., Nature, 520:186-191 (2015)).
[0082] A gene editing system designed to remove or replace one or more methylation sites of a promoter that directs expression of one or more myokine polypeptides can be designed to target a promoter that directs expression of any appropriate myokine described herein. In some cases, a gene editing system designed to remove or replace one or more methylation sites of a promoter that directs expression of one or more myokine polypeptides can target a promoter that directs expression of a Klotho polypeptide (e.g., a promoter that directs expression of an α-Klotho polypeptide such as a Klotho promoter).
[0083] Any appropriate method can be used to deliver a gene editing system (e.g., a CRISPR/Cas9 system) described herein or nucleic acid encoding a gene editing system described herein to a cell (e.g., a cell within a mammal). For example, a vector (e.g., a viral vector) can be used to deliver nucleic acid encoding a CRISPR/Cas9 system described herein to cells within a mammal (e.g. a human). In some cases, a single vector can be designed to deliver both a nucleic acid encoding the Cas9 component (e.g., an saCas9) and the targeting guide RNA of a CRISPR/Cas9 system.
[0084] In some cases, a demethylation agent that promotes expression of a myokine polypeptide (e.g., a Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment to treat the mammal as described herein. Examples of such demethylation agents that can be administered to a mammal having, or at risk for developing, an age-related impairment to treat the mammal include, without limitation, rhein (see, e.g., Zhang et al., Kidney Int., 91(1):144 (2017)) and N-(2-chlorophenyl)-1H-indole-3-caboxamide (see, e.g., Jung et al., Oncotarget, 8(29):46745-46755 (2017)).
[0085] In some cases, treating a mammal having, or at risk for developing, an age-related impairment as described herein (e.g., by increasing the level of one or more myokine polypeptides such as Klotho polypeptides in one or more cells within the mammal) can be effective to restore the healing capacity of an aged cell (e.g., an aged skeletal muscle cell) within a mammal. For example, increasing the level one or more myokine polypeptides in one or more cells within a mammal can be effective to promote healing of an aged cell (e.g., an aged skeletal muscle cell) within a mammal after an injury (e.g., an acute injury). When an aged cell is a muscle cell having damaged myofibers, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to induce myofiber regeneration (e.g., functional myofiber regeneration) in the aged muscle cell. When an aged cell is a muscle cell having damaged myofibers, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to restore the muscle cell myofiber architecture in the aged muscle cell.
[0086] In some cases, treating a mammal having, or at risk for developing, an age-related impairment as described herein (e.g., by increasing the level of one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within the mammal) can be effective to reduce cellular senescence. For example, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to reduce levels of one or more senescence markers (e.g., p16.sup.Ink4a polypeptides, p21.sup.Cip1 polypeptides, p53, H2AX, and/or SAHF) in a cell within the mammal. Any appropriate method can be used to determine a level of one or more senescence markers expressed by cells within a mammal. Examples of methods that can be used to determine a level of senescence marker expression include, without limitation, western blotting techniques, ELISA, real-time PCR, immunofluorescence, and flow cytometry.
[0087] In some cases, treating a mammal having, or at risk for developing, an age-related impairment as described herein (e.g., by increasing the level of one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within the mammal) can be effective to increase cellular bioenergetics (e.g., mitochondrial bioenergetics). For example, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to decrease mtDNA damage in a cell within the mammal. For example, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to increase the oxygen consumption rate (OCR) in a cell within the mammal. In some cases, increasing the level one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within a mammal can be effective to increase the reserve capacity in a cell within the mammal.
[0088] In some cases, treating a mammal having, or at risk for developing, an age-related impairment as described herein (e.g., by increasing the level of one or more myokine polypeptides (e.g., an α-Klotho polypeptide) in one or more cells within the mammal) can be effective to induce cellular division, to reduce fibrosis, and/or to enhance muscle progenitor cell (MPC) lineage progression.
[0089] This document also provides compositions containing (a) one or more myokine polypeptides (e.g., an α-Klotho polypeptide), (b) nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), (c) exosomes containing (i) one or more myokine polypeptides (e.g., one or more Klotho polypeptides) and/or (ii) nucleic acid encoding one or more myokine polypeptides (e.g., one or more Klotho polypeptides), and/or (d) a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide). For example, one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having, or at risk for developing, an age-related impairment. For example, one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, saline, dimethyl sulfoxide (DMSO), ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat.
[0090] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be designed for oral or parenteral (including intraperitoneal, subcutaneous, intramuscular, intravenous, and intradermal) administration to a mammal (e.g., a human) having, or at risk for developing, an age-related impairment. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. In some cases, a composition including one or more myokines and/or nucleic acid encoding one or more myokines can be formulated for intraperitoenal administration (e.g., intraperitoneal injection).
[0091] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be designed for any type of release (e.g., release of the one or more myokine polypeptides, nucleic acid encoding one or more myokine polypeptides, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide from the composition) into the mammal that the composition is administered to (e.g., a mammal having, or at risk for developing, an age-related impairment). For example, a composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be designed for immediate release, slow release, or extended release of the myokine polypeptide, nucleic acid, or gene editing system.
[0092] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered locally or systemically to a mammal (e.g., a human) having, or at risk for developing, an age-related impairment. For example, a composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered systemically by intraperitoneal administration to a mammal having, or at risk for developing, an age-related impairment.
[0093] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal (e.g., a human) having, or at risk for developing, an age-related impairment in any appropriate dose(s). Effective doses can vary depending on the severity of the age-related impairment, the risk for developing an age-related impairment, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. In cases where a composition includes one or more myokine polypeptides (e.g., an α-Klotho polypeptide), an effective dose of that composition can be from about 5 picograms of myokine polypeptides per milliliter (pg/mL) liquid (e.g., saline) to about 6 μg/mL (e.g., from about 5 pg/mL to about 5 μg/mL, from about 5 pg/mL to about 5 μg/mL, from about 5 pg/mL to about 1 μg/mL, from about 5 pg/mL to about 0.5 μg/mL, from about 5 pg/mL to about 0.1 μg/mL, from about 5 pg/mL to about 0.05 μg/mL, from about 50 pg/mL to about 1 μg/mL, from about 500 pg/mL to about 1 μg/mL, from about 1 ng/mL to about 1 μg/mL, or from about 100 ng/mL to about 500 ng/mL). In some cases, a composition including a Klotho polypeptide (e.g., α-Klotho polypeptide) can be from about 100 pg/mL to about 500 pg/mL (e.g., about 324 pg/mL). In some cases where a composition includes an α-Klotho polypeptide, the composition can be administered to deliver from about 0.001.sub.11g to about 500 μg (e.g., from about 0.01 μg to about 500 μg, from about 0.05 μg to about 500 μg, from about 0.1 μg to about 500 μg, from about 1 μg to about 500 μg, from about 10 μg to about 500 μg, from about 100 μg to about 500 μg, from about 0.001 μg to about 250 μg, from about 0.001 μg to about 100 μg, from about 0.001 μg to about 50 μg, from about 0.001 μg to about 5 μg, from about 0.1 μg to about 250 μg, from about 1 μg to about 100 μg, from about 5 μg to about 50 μg, from about 10 μg to about 50 λg, or from about 10 μg to about 30 μg) of an α-Klotho polypeptide per kg body weight of a mammal (e.g., a human).
[0094] An effective amount of a composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide can be any amount that reduces the severity and/or one or more symptom of a condition being treated (e.g., an age-related impairment) without producing significant toxicity to the mammal. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, severity of the age-related impairment, and risk for developing an age-related impairment may require an increase or decrease in the actual effective amount administered.
[0095] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal (e.g., a human) having, or at risk for developing, an age-related impairment in any appropriate frequency. The frequency of administration can be any frequency that reduces the severity of the age-related impairment and/or one or more symptoms of the age-related impairment without producing significant toxicity to the mammal. For example, the frequency of administration can be from about every three days to about ten times a day, from about every other day to about five times a day, or from about one time a day to about two times a day. In some cases, the frequency of administration can be once a day. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, severity of the age-related impairment, and risk for developing an age-related impairment may require an increase or decrease in administration frequency.
[0096] A composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal (e.g., a human) having, or at risk for developing, an age-related impairment for any appropriate duration. An effective duration for administering a composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid vector encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be any duration that reduces the severity of the age-related impairment and/or one or more symptoms of the age-related impairment without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several months or years to a lifetime. In some cases, the effective duration for the treatment of an age-related impairment can range in duration from about 2 days to about a week. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, severity of the age-related impairment, and risk for developing an age-related impairment.
[0097] In some cases, one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment as the sole active ingredient. For example, one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment as the sole active ingredient used to treat an age-related impairment. In some cases, one or more myokine polypeptides (e.g., an α-Klotho polypeptide) or nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide) can be administered as the sole active ingredient to a mammal in need thereof (e.g., a mammal such as a human having, or at risk for developing, an age-related impairment).
[0098] In some cases, one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide), one or more exosomes provided herein, and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment with one or more additional agents and/or therapies. For example, one or more myokine polypeptides (e.g., an α-Klotho polypeptide) and/or nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide) can be administered to a mammal having, or at risk for developing, an age-related impairment with one or more additional agents or therapies used to treat an age-related impairment. Examples of additional agents or therapies used to treat an age-related impairment include, without limitation, senolytics, metformin, and rapamycin. In cases where one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide), and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) are used in combination with one or more additional agents or therapies, the one or more additional agents or therapies can be administered at the same time or independently. For example, a composition including one or more myokine polypeptides (e.g., an α-Klotho polypeptide), nucleic acid encoding one or more myokine polypeptides (e.g., a nucleic acid encoding an α-Klotho polypeptide), and/or a gene editing system designed to reduce or eliminate methylation of a promoter that directs expression of a myokine polypeptide (e.g., an α-Klotho polypeptide) can be administered first, and the one or more additional agents or therapies can be administered second, or vice versa.
[0099] In certain instances, a course of treatment and the severity of one or more symptoms related to the condition being treated (e.g., an age-related impairment) can be monitored. Any appropriate method can be used to determine whether or not the severity of a symptom is reduced. For example, the severity of an age-related impairment can be assessed using any appropriate methods and/or techniques and can be assessed at different time points. For example, muscle strength, muscle endurance, and/or fine motor control can be assessed to determine the severity of an age-related impairment.
[0100] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Age-Related Declines in α-Klotho Drive Progenitor Cell Mitochondrial Dysfunction and Impaired Muscle Regeneration
[0101] While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. This example shows that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupts muscle progenitor cell (MPC) lineage progression and impairs myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drives mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restores mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhances functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age (
Results
[0102] α-Klotho is Highly Expressed in Acutely Injured Skeletal Muscle and MPCs of Young Animals, but Expression is Decreased with Aging
[0103] To determine whether α-Klotho is up-regulated locally in response to an acute muscle injury, immunofluorescence analysis of α-Klotho in the skeletal muscle was performed in young (4-6 months) and aged (22-24 months) male mice under conditions of homeostasis and following a cardiotoxin-induced injury. α-Klotho was virtually undetectable in healthy, uninjured muscle, regardless of age (
[0104] Epigenetic silencing of the α-Klotho gene contributes to the impaired regenerative potential of dystrophic skeletal muscle, and a differentially methylated region (DMR) of 110 nucleotides within the Klotho promoter region was identified in the muscles of aged mdx mice (Wehling-Henricks et al., 2016 Hum Mol Genet, 25:2465-2482). Therefore, methylation levels of the DMR after injury were measured in young and aged muscle of mice. An acute injury to young muscle triggered demethylation of the DMR in the Klotho promoter three and seven days after injury (
[0105] To examine whether modifying enzymes for DNA methylation contribute to methylation changes in the Kl promoter, a chromatin immunoprecipitation (ChIP) assay was used to measure the enrichment of DNMT3a methyltransferases in the Klotho promoter region (see, e.g., Wehling-Henricks et al., 2016 Hum Mol Genet, 25:2465-2482). There was a decrease of DNMT3a binding to the Klotho promoter in the injured young muscle that, at the time points evaluated, reached a nadir three days after injury, and was increased by day 14 (
Genetic Inhibition of α-Klotho Impairs Skeletal Muscle Regeneration
[0106] To directly implicate a functional role for α-Klotho in skeletal muscle regeneration, the regenerative response to acute muscle injury in adult mice that are heterozygously deficient for Klotho (Kl+/− mice) was evaluated. Kl+/− mice displayed a significantly decreased local α-Klotho expression at the site of injury (
[0107] Given our findings of a significantly increased Klotho expression within the injured muscles of young mice (
[0108] Histological analysis revealed that knock down of α-Klotho expression resulted in a decreased number of regenerating fibers, a decrease in the percentage of myofiber area/total area, and an increased adiposity (
α-Klotho is Expressed by MuSCs and Their Progeny
[0109] α-Klotho expression in young muscle is elevated at 3 days after injury (
[0110] To determine whether MPCs secrete α-Klotho, ELISA of the conditioned media from MPCs isolated from young and old muscle was performed. Conditioned media derived from young MPCs contained significantly more α-Klotho than the conditioned media obtained from aged MPCs (
[0111] Immunohistochemical analysis of young muscle three days after injury revealed that 96.7% of MyoD+cells express α-Klotho (
[0112] It was next asked whether α-Klotho is necessary for normal MuSC lineage progression. MPCs isolated from the skeletal muscle of Kl+/− mice displayed a small, but significant, decrease in the percentage of MyoD+cells when compared to age-matched wild type counterparts. There was, however, no difference in Pax7 expression across groups (
Loss of α-Klotho Expression in MPCs Drives Cellular Senescence and Mitochondrial Dysfunction
[0113] It was investigated whether the decreased MuSC activation in α-Klotho-deficient muscles may be attributed to increased cellular senescence. To confirm an inhibitory role for α-Klotho in cellular senescence in myogenic cells, small interference RNA (siRNA) inhibition was used, which resulted in a ˜3-fold decrease in α-Klotho (
[0114] LXRepair multiplex technology (see, e.g., Garreau-Balandier et al., 2014 FEBS Lett, 588:1673-9; and Sauvaigo et al., 2004 Anal Biochem, 333:182-92) was used to evaluate the DNA base excision repair (BER) enzyme activities of OGG1 and APE1, which work on two common oxidative DNA lesions, 8-oxodG and abasic sites, respectively. When compared to scramble siRNA-treated young MPCs, there was no significant decline in base excision repair (BER) in the nucleus of cells treated with siRNA to α-Klotho (
[0115] While α-Klotho's role in cellular senescence has been demonstrated in multiple systems, the mechanisms underlying this role are incompletely understood. The possibility that α-Klotho may regulate MPC mitochondrial structure and function was evaluated. Using an antibody against the mitochondrial membrane protein, Tom20, no difference in mitochondrial morphology was observed, as determined by sphericity, the number of mitochondria per cell, or mitochondrial volume according to age or α-Klotho levels (
[0116] In light of the loss of mitochondrial ultrastructural resulting from decreased α-Klotho expression, we next evaluated whether loss of α-Klotho drives MPC mitochondrial dysfunction. The bioenergetic profiles of young and aged MPCs were studied using a Seahorse XFe96 Flux analyzer, which measures oxygen consumption rate (OCR), a measure of oxidative phosphorylation. OCR was measured again after injection of oligomycin, FCCP, 2-deoxyglucose (2-DG), and rotenone. These data demonstrate that, when normalized to total number of cells, MPCs from older animals display dramatically decreased levels of basal OCR as compared to young counterparts (
[0117] mtDNA integrity was next examined in MPCs isolated from young or aged mice, using a qPCR-based assay. The method used is based on the principle that a wide variety of types of DNA damage have the propensity to block DNA polymerase progression (see, e.g., Furda et al., 2012 DNA Repair (Amst), 11:684-92). Therefore, this assay detects numerous kinds of base DNA damage or DNA repair intermediates such as abasic sites, as well as single and double DNA strand breaks.
[0118] There was no difference in steady-state mtDNA copy number across groups (
α-Klotho Preserves Mitochondrial Function through Maintenance of Mitochondrial Ultrastructure
[0119] The disrupted mitochondrial cristae structure within cells displaying decreased α-Klotho expression (
[0120] Cardiolipin is an anionic phospholipid that is confined almost exclusively to the inner mitochondrial membrane where it is synthesized. It was found that cardiolipin content is significantly depleted in MPCs isolated from Kl+/− mice, as compared to wild type counterparts (
Supplementation with α-Klotho Restores MPC Bioenergetic Profile In Vitro and Enhances Muscle Regeneration In Vivo
[0121] Given the established hormonal role of α-Klotho, it was next investigated whether supplementation of α-Klotho may restore mitochondrial function in aged MPCs. It was found that when MPCs isolated from aged skeletal muscle were cultured in the presence of recombinant α-Klotho for 48 hours, the aged mitochondrial phenotype was improved, as determined by decreased mtDNA damage, an increased OCR, and increased reserve capacity (
[0122] These encouraging in vitro findings led us to next probe whether α-Klotho supplementation may enhance skeletal muscle regeneration in vivo. To do this, α-Klotho was administered to aged mice via osmotic pump delivery. Osmotic pumps were implanted three days prior to injury and were maintained for 14 days post-injury. At the dose tested, a significant increase in local α-Klotho was observed within the injured muscle areas (
[0123] Given that osmotic pump administration delivers α-Klotho continuously, it was next tested whether the timing of α-Klotho administration may be critical for functional tissue regeneration. To do this, daily intraperitoneal injections of recombinant α-Klotho to aged mice either from 1-3 days post injury (dpi) or from 3-5 dpi (i.e. the time point at which we found Klotho to be highly expressed in young muscle) were performed (
[0124] Taken together, the data suggest that α-Klotho is required for an adequate regenerative response to an acute injury, and that supplementation with α-Klotho via the circulatory system promotes MuSC commitment and myofiber regeneration in aged mice when administered at the appropriate time point. These findings implicate declines in this longevity protein as a contributor to a defective muscle regenerative response with aging and raise the possibility of systemic administration of α-Klotho as a therapeutic approach to promote the healing of aged skeletal muscle after injury.
Methods
Animals
[0125] C57BL/6 young (4-6 months) and old (22-24 months) mice were received from the Jackson laboratories and NIA rodent colony, respectively. Kl+/− mice were obtained from MMRRC, UC Davis and were genotyped prior to inclusion in the studies. All animals were ear-tagged, randomly assigned to intervention group, and compared to age-matched littermate controls whenever possible. Mice were evaluated prior to inclusion in the study, and animals with obvious health problems were eliminated. Animal experiments were repeated across a minimum of two separate cohorts of the experimental groups. All primary endpoints were prospectively selected prior to analyses and investigators performing endpoint analysis were blinded to the experimental group whenever possible.
Animal Injury Model and Histological Analysis of Muscle Regeneration
[0126] Wild-type male C57BL/6 young, Kl+/− mice, or old mice received injuries to bilateral Tibialis Anterior (TA) muscles via an intramuscular injection of cardiotoxin (10 μL of 1 mg/mL). Fourteen days following the injury, TAs were harvested for histological analysis of α-Klotho, fibrosis (picrosirius red), degenerating myofibers (IgG) and myofiber regeneration (laminin). Second Harmonic Generation (SHG) imaging was performed on isolated TA muscles treated with a non-targeting control or lentiviral knockdown of α-Klotho, as well as pump-administered animals in order to visualize collagen and myofibers within the muscle 14 days post injury, as described elsewhere (see, e.g., Zhang et al., 2016 Stem Cells 34:732-742).
Primary Muscle Cell Isolation
[0127] MPCs were isolated from young, Kl+−, and aged mice, as described elsewhere (see, e.g., Zhang et al., 2016 Stem Cells 34:732-742). MuSCs were sorted using FACS for surface markers CD31−, CD45−, Sca1− and VCAM+ as described elsewhere (see, e.g., Cheung et al., 2012 Nature, 482:524-8). A modified protocol was used to isolate MuSCs and FAPs as CD31−, CD45−, α-7 integrin+ for MuSCs and CD31−, CD45− and α-7 integrin- for FAPs as described elsewhere (see, e.g., Yi and Rossi, 2011 J Vis Exp. 16:2476).
Primary Muscle Cell Imaging
[0128] Immunofluorescence staining (α-Klotho, Tom20 (mitochondrial marker), ki67, MyoD, Pax7 and HMGB1) and senescence-associated beta-galactosidase staining was performed in isolated cells. Transmission electron microscopy of fixed cells was performed, as described elsewhere (see, e.g., Zhang et al., 2016 Stem Cells 34:732-742). Structured illumination microscopy was performed in young and old cells stained for α-Klotho and DAPI.
ELISA
[0129] The levels of α-Klotho protein were measured by a colorimetric sandwich enzyme immunoassay (SEH757Mu, Cloud-Clone Corp), according to manufacturer's instructions. Each sample was measured in duplicate.
Hanging-Wire Test
[0130] Strength endurance was tested using the hang-wire test as described elsewhere (see, e.g., Aartsma-Rus et al., J. Vis. Exp 85:51303 (2014)). The Hang Impulse (HI) score was calculated as bodyweight (grams)×time hung (seconds). Male mice were used for all testing using C57B1/6 mice. Wild type and Kl+/− were females for testing.
Inhibition of and Supplementation with α-Klotho
[0131] MPCs were treated with 25 nmol of silencing RNA (siRNA) to α-Klotho (GE Dharmacon, Product no.SO2462181G) for 48 hours. As a control, young MPCs were treated with a non-targeting (scramble) siRNA. Aged MPCs were treated with 0.05μg/mL exogenous α-Klotho (R & D systems, Product no. aa 34-981), added to the culture media for 48 hours.
Epigenetic Regulation of α-Klotho
[0132] At baseline, three, seven, and fourteen days after injury, TAs were snap frozen using liquid nitrogen for gene expression, methylation specific PCR (MSPCR), and chromatin immunoprecipitation (ChIP) analysis, essentially as described elsewhere (see, e.g., Lin et al., 2016 Am J Respir Cell Mol Biol, 54:241-9).
Analysis of MPC Bioenergetics and Mitochondrial DNA Damage
[0133] Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real time using a Seahorse XFe96 Extracellular Flux Analyzer (Billerica, Mass.) as described elsewhere (see, e.g., de Moura and Van Houten, 2014 Methods Mol Biol, 1105:589-602). The basal OCR was measured by averaging the OCR values before treating the cells with oligomycin. Total reserve capacity was calculated by the differences of OCR between treatment with FCCP and 2DG and basal values. Mitochondrial DNA damage was quantified as described elsewhere (see, e.g., Sanders et al., 2014 Toxicol Sci, 142:395-402; and Sanders et al., 2014 Neurobiol Dis, 62:381-6).
SS-31 Administration
[0134] Isotonic saline or SS-31 (3 mg/kg dissolved in saline at 0.3 mg/mL) was administered daily via an i.p. injection to wild-type and Kl+/− animals for the entire duration of injury. For in vitro experiments, 100 nM of SS-31 was administered to MPCs isolated from Kl+/− animals for 48 hours. Dosing was based on studies demonstrating the effectiveness in a mouse model of chronic cardiomyopathy (see, e.g., Dai et al., 2011 Am Coll Cardiol, 58:73-82) as well as in vitro dose ranging studies performed in C2C12s to evaluate inhibition of stress-induced mitochondrial membrane hyperpolarization and ROS generation.
In Vivo Lentiviral Knockdown of α-Klotho
[0135] In-vivo α-Klotho knockdown was done using lentiviral vectors for a SMARTpool of 2.0×10.sup.5 TU/TA or 3.82×10.sup.6 TU/TA shRNA to α-Klotho per TA muscle. Given that there was no significant difference in the local α-Klotho expression between the two treatment groups, samples across the two treatment groups were pooled for analysis. Control animals received equal volumes of empty lentiviral vector. Knockdown was maintained for three weeks, after which time bilateral TAs were injured. Histology or SHG imaging was performed 14 days after injury.
Supplementation of α-Klotho In Vivo
[0136] Mini osmotic pumps containing either saline or α-Klotho (324 pg/ml in saline vehicle) were inserted subcutaneously into aged mice. After 2 days, bilateral TA muscles were injured by intramuscular CTX injection (as above). Osmotic pumps remained implanted until euthanasia 14 days post injury. Isotonic saline or α-Klotho (10 μg/kg body weight) was administered to aged animals via daily intraperitoneal injections over days 1-3 post-injury or 3-5 days post-injury. The TAs were then harvested 14 days post injury and preserved for histology or SHG analysis. Blood serum was also collected to evaluate circulating α-Klotho levels via ELISA. The activity of α-Klotho was confirmed as described elsewhere (see, e.g., Shalhoub et al., 2011 Calcif Tissue Int, 89:140-50).
Example 2: Exosome-Mediated Delivery of Klotho to Improve Muscle and Brain Function
[0137] Isolation of Exosomes from Brain, Plasma and CSF
[0138] Exosomes were isolated from the brain tissue using a method described elsewhere (see, e.g., Vella et al., 2017 J Extracell Vesicles. 6:1348885). Brain slices from young WT mice (0.5-1 g) were enzymatically digested (collagenase type III in Hybernate E). Exosomes were isolated from CSF (15 μl sample) and plasma (250 μl sample) using a method described elsewhere (see, e.g., Filant et al., Methods Mol Biol. 1740:43-57). Exosomes from these three compartments were isolated using density gradient ultracentrifugation (sucrose) and analyzed using Zetasizer Nano ZS to determine size distribution of each fraction. In the exosome fraction (F2) vesicles detected had average size of 70 nm (
Exosomal Delivery of Klotho
[0139] Klotho mRNA is Detected in Exosomes Isolated from Human Serum; Expression is Generally Decreased in Individuals with Coronary Heart Disease
[0140] RNAseq data which is housed in a public repository of experimentally validated RNA-seq data analyses from the published literature of human blood exosomes were accessed. Analysis of archived data revealed that Klotho mRNA was detectable in exosomes isolated from human sera (
Klotho Protein is Detected in Exosomes Isolated from Brain, Cerebrospinal Fluid (CSF) and Mouse Serum.
[0141] Brain slices from young wild type mice (0.5-1 g) were enzymatically digested using collagenase type III in Hybernate E. To isolate exosomes from CSF (15 μL sample) and for plasma (250 μL sample), a method described elsewhere was used (Filant et al., Protocol Exchange (2017)). Exosomes were isolated using density gradient ultracentrifugation (sucrose) and analyzed using Zetasizer Nano ZS to determine size distribution of each fraction. In the exosome fraction (F2), the average size of the vesicles was 70 nm (
Neuromuscular Electrical Stimulation Increases Klotho Protein Expression in Murine Circulating Exosomes.
[0142] Next, young (4-6 months) mice were exposed to a neuromuscular electrical stimulation (NMES) protocol as described elsewhere (Ambrosio et al., J. Vis. Exp., e3914 (2012)). Exosomes were then isolated using size-exclusion chromatography from NMES stimulated as well as age- and sex-matched sedentary control mice. 2 weeks of NMES increased the amount of protein content within circulating exosomes, as determined by immunofluorescence (
Example 3: Extracellular Vesicle Delivery of Klotho Transcripts Rejuvenates Aged Stem Cell Progeny
[0143] This Example shows that depletion of EVs eliminated the beneficial effect of young serum on the bioenergetics of target MuSC progeny, and that the impact of EVs on target cell mitochondrial function was a result of Klotho mRNA transfer. Machine learning classifiers further revealed that aging disrupts EV population heterogeneity through a selective loss of CD63.sup.+ extracellular vesicles, which preferentially contain Klotho mRNAs. In vivo, it was shown that Klotho mRNA content within EVs supported muscle regeneration after acute injury (
Results
Circulating Extracellular Vesicles Modulate the Bioenergetics of Target Cells in an Age-Dependent Manner
[0144] It was found that expression of MyoD, a master regulator of myogenesis, was increased in aged MuSC progeny when cultured in the presence of young serum (
[0145] To determine whether EVs contribute to the observed effects of young serum on muscle progenitor cell mitochondrial function, young and aged serum were depleted of circulating EVs (
Aging Shifts Circulating EV Population Heterogeneity Through Preferential Loss of CD63.SUP.+ Extracellular Vesicles
[0146] The above findings demonstrate that circulating EVs may regulate the bioenergetics of target muscle progenitor cells, but that aging disrupts this information flow. The transfer of information between EVs and their target cells is mediated by a wide range of EV cargoes, including cytosolic proteins, membrane proteins, mRNAs, noncoding RNAs, and DNA. However, age-associated changes in the circulating EV structure and cargo have not yet been thoroughly investigated. Therefore, an in-depth analysis of young and aged EVs that were isolated using size-exclusion chromatography (qEVsingle, iZON columns) was performed. Nanoparticle tracking analysis using NanoSight apparatus (Product# NS300) confirmed that the size of isolated EVs was less than 200 nm in diameter (
[0147] NanoSight quantifies the total concentration of nanoparticles in a non-discriminant manner to include microvesicles, exosomes, and apoptotic bodies. Multispectral flow cytometry imaging (IFC; ImageStream) was used to classify nanoparticles according to differential expression of the three EV markers: CD63, CD81, and CD9. In order to phenotypically resolve the EV signal using IFC, the Fischer's discriminant ratio was utilized as a class separability criterion, followed by feature selection and gating according to intensity-based clustering (
TABLE-US-00001 TABLE 1 ML Method Accuracy AUC Precision F1 Score Recall LogLoss Nearest Neighbors 0.89885 0.972382 0.897171 0.89683 0.896849 0.214144 Decision Tree 0.88152 0.965188 0.88156 0.881517 0.881521 0.235735 Random Forest 0.880687 0.967128 0.880735 0.880683 0.880688 0.292276 Neural Network 0.733378 0.826779 0.73501 0.732918 0.733385 0.500638 Gradient Boosted Trees 0.696717 0.781014 0.69673 0.696712 0.696717 0.573117 Naive Bayes 0.653391 0.71043 0.6534 0.653387 0.653392 0.622839 Logistic Regression 0.563073 0.71043 0.6534 0.653387 0.653392 0.622839 Metrics used to evaluate classifier performance without bootstrapping of data. The training set (⅔ of the entire EV population) contained the known age-class output. The computational model learned on this dataset in order to be generalized to the test dataset.
[0148] To address the physiological relevance of age-related changes in EV composition, the direct effect of young or aged EVs on target muscle progenitor cell responses was evaluated. Consistent with serum co-culture experiments described above, it was found that aged cells cultured in the presence of young EVs, but not old EVs, displayed increased MyoD expression and mitochondrial cardiolipin content (
EV Nucleic Acid Content is Compromised with Aging
[0149] To better understand whether age-related alterations in EV cargo underlie alterations in target cell responses, Raman spectroscopy analysis of young and aged EVs was performed. This method allows for bulk characterization of EV biochemical composition according to light scattering properties. Qualitative EV differences as a function of age were evaluated by subtracting the aged EV spectra from the young, and the resulting Raman spectral peaks were assigned to functional chemical groups, as described elsewhere (see, e.g., Movasaghi et al., Applied Spectroscopy Reviews 42 (2007)). There were no appreciable differences in the protein content of EVs according to age, which was further confirmed by a bicinchoninic acid assay (
Klotho Transcripts are Abundant in Young EVs, but Their Content is Decreased with Age
[0150] It was next sought to identify specific alterations in the EV cargo that may contribute to changes in target cell responses. The data presented thus far suggest that aging disrupts EV composition and the transfer of mitochondrially-targeted information to recipient MuSC progeny. It was examined whether the transfer of EV cargo alters Klotho protein within recipient cells, thereby regulating mitochondrial function.
[0151] To test this, aged muscle progenitor cells were cultured either in the presence or absence of young serum-derived EVs. Immunofluorescence imaging revealed that Klotho protein in target cells increased by ˜40% when cells were exposed to young EVs (
[0152] To more directly implicate Klotho signals originating from the EVs, the impact of EVs isolated from Klotho.sup.−/− mice on Klotho protein levels in target muscle progenitors was tested. It was found that EVs from Klotho.sup.−/− mice were toxic to the cells. Cells were then cultured in the presence of EVs isolated from the serum of either Klotho.sup.+/− mice or age- and sex-matched wild type control mice. The increased Klotho protein levels observed when aged cells were incubated with young Klotho.sup.+/+ EVs was blunted when the EVs were isolated from young Klotho.sup.+/− mice (
[0153] Given these data, it was examined whether EVs carry and transmit the Klotho protein in an age-dependent manner. The presence of Klotho protein within serum-derived EVs appears unknown. Surface Plasmon Resonance imaging (SPRi) analysis, however, revealed that circulating EVs do indeed contain Klotho protein on their membranes (
[0154] The Raman spectroscopy-based finding that aged EVs display a marked decline in nucleic acid content prompted further analysis into whether the increase in Klotho protein by target muscle progenitors may be a result of the transfer of genetically encoded information by EVs. mRNAs packaged within the EVs are functional and capable of being translated when in the presence of the requisite protein machinery of the target cells. To detect and quantify Klotho mRNA within peripheral EVs, a set of Klotho oligonucleotides was designed to probe for Klotho transcripts. IFC revealed that young EVs contained abundant levels of Klotho mRNAs, but that Klotho mRNA content was significantly decreased in aged EVs (
[0155] To more directly test whether EV-derived Klotho mRNA is a source of Klotho protein within target cells, whether treatment of young EVs with a small interfering RNA to Klotho would abate the Klotho response of recipient progenitors was evaluated. Digital PCR confirmed a decrease in Klotho mRNA of approximately 20% when EVs were treated with siRNA to Klotho (
[0156] To rule out the possibility that the effect of EVs on progenitor cell Klotho protein expression may result from the transmission of some transcriptional regulator that promotes endogenous Klotho mRNA expression in target cells, young EVs were delivered to muscle progenitor cells isolated from Klotho.sup.−/− mice, which lack a functional endogenous Klotho locus. Just as was observed in wild type cells, Klotho.sup.−/− cells cultured in the presence of young EVs displayed a significant increase in Klotho protein, and the effect was blunted following treatment with siRNA to Klotho (
Klotho mRNA Cargoes of Circulating EVs Enhance Muscle Functional Recovery after Injury
[0157] Next, a series of studies was designed to evaluate whether EVs isolated from the circulation may contribute, upon transplantation, to the skeletal muscle regenerative cascade after an acute injury. To induce a muscle injury, aged animals received cardiotoxin injections to bilateral tibialis anterior muscles. The average functional defect one day after injury was approximately 50% of baseline levels (
Methods
Serum and Muscle Progenitor Cell Isolation
[0158] Serum of young and aged C57/BL6 mice (obtained from Jackson laboratories and NIA Rodent colony, respectively) as well as Klotho.sup.+/+, Klotho.sup.+/− and Klotho.sup.−/− mice (original breeders obtained from MMRCC, UC Davis) was obtained from animals using a cardiac puncture. Skeletal muscle progenitor cells were isolated from aged C57/BL6 (22-24 months) and Klotho.sup.−/− male mice (8 weeks) as described elsewhere (see, e.g., Sahu et al., Nat. Commun., 9:4859 (2018)).
Immunofluorescence Imaging
[0159] Immunofluorescence staining for Klotho (R&D systems, MAB1819) and MyoD (SCBT, sc-760) as well as nonyl acridine orange (NAO) staining (Thermofisher, A1372) for cardolipin content was performed on aged cells across experimental groups. Muscle sections were analyzed for fiber cross-sectional area using an antibody against Laminin (Abcam, ab11575). Imaging was performed at 20× magnification on Zeiss-Axiovision microscope.
Analysis of Cellular Bioenergetics
[0160] Oxygen consumption rate (OCR) was measured in real time using a Seahorse XFe96 Extracellular Flux Analyzer (Billerica, Mass.) as described elsewhere (see, e.g., de Moura et al., Methods Mol. Biol., 1105:589-602 (2014)).
EV Isolation and Characterization
[0161] EVs were isolated from serum of young, aged, Klotho.sup.+/+ and Klotho.sup.+/− animals using size-exclusion chromatography (qEVsingle-35 nm iZON columns) according to manufacturer's instructions. The EVs were characterized for size by Nanoparticle Tracking Analysis on NanoSight NS300. EVs were then characterized for CD63 (SCBT 5275), CD81 (SCBT 23962), and CD9 (SCBT 13118) markers using multispectral flow cytometry based ImageStream analysis. EV marker CD63 was further confirmed using SPRi and in-cell western blot.
ImageStream Imaging and PrimeFlow™ RNA Assay
[0162] EVs isolated from young and aged serum were analyzed using Amnis® ImageStream®XMark II (Luminex Corporation). First, samples were processed with filtered sheath buffers to ensure the removal of big particulates and debris (≥1 μm). Flow cytometry was then performed using a 60× objective at a resolution of 0.3 μm.sup.2/pixel. Both brightfield and fluorescent images of the EVs were captured using the INSPIRE® software with the highest resolution (sensitivity) and lowest speed. An integrative technical computing framework with multiple machine learning modules and statistical analyses was utilized using R/Python and Wolfram programming languages to analyze the EV signals.
[0163] PrimeFlow™ was performed according to the manufacturer's instructions. Two standard 20bDNAs Mouse Klotho oligos probe sets (VB1-6001084 (Part No. 6003837) and VB10-6001085 (Part No. 6003838)) tagged with Type 1 Alexafluor (AF)647 and Type 10 Alexafluor (AF) AF568 dyes, respectively were utilized. mRNA expression was reported based on the mean fluorescence intensity (MFI) at the single EV resolution.
SPR Imaging and Analysis
[0164] EVs were injected into the flow cell of the SPRi instrument XelPleX (Horiba Scientific SAS). The EVs were then injected over a gold chip (SPRi-Biochip, Palaiseau, France) onto which antibodies against CD63 and Klotho were spotted using a micro-spotter (SPRi Arrayer, Horiba). EzSuite software and OriginLab software were used to analyze the collected sensograms.
Raman Spectroscopy Young and aged EVs isolated by size-exclusion chromatography were concentrated by an ultracentrifugation step (100,000 g×70 minutes). These EVs were then analyzed by means of Raman spectroscopy (LabRAM, Horiba Jobin Yvon S. A. S. Lille, France) as described elsewhere (see, e.g., Gualerzi et al., Sci. Rep., 7:9820 (2017)).
ELISA
[0165] Aged muscle progenitor cells were cultured at 10,000 cells per well of an 8-well chamber slide, for 24 hours prior to treatment with young EVs. The conditioned media was collected 48 hours post-administration, and levels of Klotho protein in conditioned media were measured by a colorimetric sandwich enzyme immunoassay (SEH757Mu, Cloud-Clone Corp), according to manufacturer's instructions. The protein concentration was then normalized to total number of cells per well.
Functional and Histological Analysis of Muscle Regeneration of Injured Animals
[0166] Wild-type male C57BL/6 (22-24 months) and Klotho.sup.+/− mice (4-7 months) mice received injuries to bilateral Tibialis Anterior (TA) muscles via an intramuscular (i.m.) injection of cardiotoxin (10 μL of 1 mg/mL). Three days post-injury, the animals received 20-30 μL of bilateral i.m. injections of EVs, and in situ contractile testing was performed two weeks after injury as described elsewhere (see, e.g., Zhang et al., Stem Cells 34:732-742 (2016)). The overall muscle endurance of mice was tested at one- and 13-days post injury using a modified hanging-grid test (see, e.g., Sahu et al., Nat. Commun., 9:4859 (2018); and Aartsma-Rus et al., J. Vis. Exp., 85:e51303 (2014)). The hang time for each mouse was normalized to mouse weight. TA muscles were harvested for histological analysis of myofiber regeneration using an antibody against Laminin (Abeam, ab11575). All animals were randomly assigned to intervention group based on their baseline hang-impulse scores and were compared to age-matched littermate controls whenever possible.
Steps to Ensure Rigor
[0167] For all experiments, investigators performing endpoint analyses were blinded to the treatment group. To do this, animals were ear-tagged for in vivo analyses, and samples were number-coded. Animals with obvious health problems were eliminated prior to inclusion in the study. All animals meeting criteria for inclusion were then randomly assigned to treatment groups.
Code Availability
[0168] The computational code used to perform machine learning based analyses on EVs are available online at github.com/SelfHorizonsWork/Nature-Extracellular-vesicle-delivery-of-Klotho-transcripts-rejuvenates-aged-stem-cell-progeny. To generate PCAs, the OriginLab plugin called “Principal Component Analysis for Spectroscopy” was used.
Example 4: Engineering EVs with Synthetic Klotho mRNA
[0169] EVs with synthetic Klotho mRNA were engineered. Briefly, EVs were transfected with the synthetic mRNA sequences (Klotho oligos) using Exo-Fect™ Exosome Transfection Reagent from System Biosciences (Cat. No. EXFT-10A1). The transfection was done as per manufacturer's suggested protocol. Briefly, ˜e9 EVs received 150 μL exofect solution and 1 μg amount of synthetic mRNA. This solution was mixed well by flicking the tube 3 times. Samples were incubated at 37° C. for 10 minutes and then put on ice for 30 minutes to stop the reaction. Samples were then centrifuged at 13k-14k rpm. The supernatant was removed, and sample was resuspended in media for in vitro application. 10k aged muscle progenitors were incubated with loaded EVs for 48 hours (
OTHER EMBODIMENTS
[0170] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.