Methods and compositions for cell and tissue rejuvenation

Abstract

The present disclosure provides compositions, methods and kits for the rejuvenation of target cells. In some aspects, the compositions, methods and kits comprise mRNAs the promote the expression of TERT and/or TERC.

Claims

1. A composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a biologically active exogenous human telomerase reverse transcriptase (TERT) having telomere elongation activity; and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least a portion of the at least one DNA targeting polypeptide increases transcription of endogenous human telomerase RNA component (TERC) compared to untreated cells wherein none of the DNA-targeting polypeptides target an endogenous TERT gene, wherein the DNA-targeting polypeptide does not target an endogenous TERT gene, and wherein the combination of a) and b) increases telomere length compared to untreated cells.

2. The composition of claim 1, wherein the at least one first polynucleotide molecule comprises: i) an mRNA molecule encoding at least a biologically active human TERT having elongation activity; or ii) a plasmid comprising a nucleic acid sequence encoding at least a biologically active human TERT operably linked to at least one promoter to drive detectable expression of the at least one biologically active human TERT.

3. The composition of claim 1, wherein the at least one second polynucleotide molecule comprises: i) an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide; or ii) a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter to drive detectable expression of the at least one portion of the at least one DNA targeting polypeptide.

4. The composition of claim 1, wherein the DNA targeting polypeptide comprises at least one CRISPR-associated protein 9 (Cas9) molecule, at least one Cas9 variant that remains functionally active, or at least one Cas9 ortholog thereof that remains functionally active, at least one Transcription Activator-Like Effector (TALE) molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof.

5. The composition of claim 1, wherein the DNA targeting polypeptide comprises at least one transactivation molecule.

6. The composition of claim 1, wherein when the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant that remains functionally active, or at least one Cas9 ortholog thereof, the composition further comprises at least one guide RNA (gRNA).

7. The composition of claim 5, wherein the transactivation molecule comprises at least one single guide RNA MS2 bacteriophage (sgRNA-MS2) molecule, wherein the at least one sgRNA-MS2 molecule comprises a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least one MS2 RNA aptamer.

8. The composition of claim 1, further comprising: i) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene; or ii) at least one plasmid comprising at least one nucleic acid sequence encoding at least one gRNA operably linked to at least one promoter to drive detectable expression of the at least one species gRNA.

9. The composition of claim 1, wherein the nucleic acid sequence encoding at least a portion of the at least one DNA targeting polypeptide in b) comprises at least one modified mRNA molecule and wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and further comprising: c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA is selected from the group consisting of SEQ ID nos. 1-1276.

10. The composition of claim 1, further comprising at least one polynucleotide encoding at least one rejuvenating factor.

11. The composition of claim 1, wherein the composition is packaged or encoded in at least one viral particle, at least one exosome, at least one microvesicle, at least one liposome, or at least one nanoparticle.

12. A method of rejuvenating at least one cell, the method comprising contacting the at least one cell in need of rejuvenation with the composition of claim 1.

13. A method of treating, reducing the risk of onset of, or preventing a health condition in a subject comprising: a) contacting at least one cell in vitro with the composition of claim 1; b) expanding the at least one cell in vitro to produce a plurality of rejuvenated cells; and c) administering a therapeutically effective amount of the plurality of rejuvenated cells to the subject in need of cell therapy and treating, reducing onset of, or preventing the health condition in the subject.

14. A method for rejuvenating at least one cell in a subject comprising administering to the subject a therapeutically effective amount of the composition of claim 1.

15. The composition of claim 1, further comprising a pharmaceutically acceptable excipient.

16. A kit comprising the composition of claim 1, and at least one container.

17. A method for preparing a composition of claim 1 comprising combining a) and b) into a medium.

18. The composition of claim 1, wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant that remains functionally active, or the at least one Cas9 ortholog molecule thereof that remains functionally active and the at least one Cas9 molecule, the at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof comprises at least one of eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D11 35E), Cas9 (QQR1), SaCas9 (KKH, Nme1Cas9, Nme2Cas9, Nme3Cas9, Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Cas (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

19. The composition of claim 4, wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof and the at least one Cas9 molecule, the at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof comprises at least one of SpCas9, SaCas9, SpyFi Cas9, Cpf1 and xCas9, or variant or ortholog molecule thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

(2) FIG. 1 is a schematic of a DNA-targeting molecule of the present disclosure binding upstream of an endogenous hTERC locus. In this non-limiting example, the DNA-targeting molecule comprises dCas9 and a transactivation molecule, wherein the transactivation molecule is a VP64-P65-Rta (VPR) molecule.

(3) FIG. 2 is a schematic overview of a treatment method and/or a method of producing an in vitro tissue of the present disclosure

(4) FIG. 3 is a schematic overview of a method of producing a plurality of rejuvenated edited cells of the present disclosure.

(5) FIG. 4 is a chart showing the hTERC transcript level in various cell types.

(6) FIG. 5 is a chart showing the level of human TERC RNA in F50 cells transfected with compositions of the present disclosure.

(7) FIG. 6 is a series of charts showing the level of human TERC RNA in HEKn cells (left) and human Mesenchymal Stem/Stromal Cells (hMSCs) (right) transfected with compositions of the present disclosure (+dCas9VPR+gmix) as compared to non-transfected HEKn cells, non-transfected hMSC cells and F50-derived induced pluripotent stem cells.

(8) FIG. 7 is a series of charts showing the level of human TERC RNA in F50 cells (left) and hMSCs (right) transfected with compositions of the present disclosure lacking guide RNA (+dCas9VPR (no guide)).

(9) FIG. 8 is schematic overview of one of the transfection regimes of the present disclosure.

(10) FIG. 9 is a chart showing the total population doubling of senescent F50S cells transfected with compositions of the present disclosure.

(11) FIG. 10 is a schematic overview of an alternative transfection regime of the present disclosure.

(12) FIG. 11 is a schematic overview of another transfection regime of the present disclosure.

(13) FIG. 12 is a chart showing the relative telomere length in senescent F50S cells transfected with compositions of the present disclosure (+TERT+dCas9VPR/gmix) as compared to non-transfected F50S cells and F50-derived induced pluripotent stem cells.

(14) FIG. 13 is a chart showing the relative telomere length in F50 cells transfected with compositions of the present disclosure (+hTERT+dCas9VPR/gmix) as compared to non-transfected F50 cells and F50-derived induced pluripotent stem cells.

(15) FIG. 14 is a series of charts showing the relative telomere length in HEKn cells (left) and hMSCs (right) transfected with compositions of the present disclosure (+TERT+dCas9VPR/gmix) as compared to non-transfected HEKn cells, non-transfected hMSCs and F50-derived induced pluripotent stem cells.

(16) FIG. 15 is a series of charts showing the relative amount of mitochondrial DNA in F50 cells (left), HEKn cells (middle) and hMSCs (right) transfected with compositions of the present disclosure (+TERT+dCas9VPR/gmix) as compared to non-transfected F50 cells, non-transfected HEKn cells and non-transfected hMSCs.

(17) FIG. 16 is a gel image of the results of a telomerase activity assay in F50 cells transfected with various compositions of the present disclosure (F50+TERT and F50+TERT+dCas9VPR/gmix) as well as non-transfected F50 cells and F50-derived induced pluripotent stem cells.

(18) FIG. 17 is a series of representative microscopy images of adult human primary fibroblasts expanded from single cells that were not transfected (top two rows) or transfected with compositions of the present disclosure (+hTERT/dCas9VPR+gRNA; bottom two rows).

(19) FIG. 18 is a schematic overview of the transendothelial migration (TEM) assay.

(20) FIG. 19 is chart showing the migration activity of hMSCs transfected with compositions of the present disclosure as measured using the TEM assay.

(21) FIG. 20 is series chart showing the oxidation level of thiol groups detected in selected proteins in senescent hMSCs transfected with compositions of the present disclosure as compared to non-transfected young low passage and senescent high passage hMSCs.

(22) FIG. 21 is a series chart showing the degree of methylation at 9 senescence-associated DNA methylation sites in senescent cells of different types transfected with compositions of the present disclosure as compared to non-transfected young low passage and senescent high passage cells.

DETAILED DESCRIPTION OF THE INVENTION

(23) Telomeres comprise repetitive DNA sequences at the ends of linear chromosomes that, when sufficiently long, allow each chromosome end to form a loop that protects the ends from acting as double-stranded or single-stranded DNA breaks. Telomeres shorten over time, due in part to oxidative damage and incomplete DNA replication, eventually leading to critically short telomeres unable to form the protective loop, exposure of the chromosome ends, chromosome-chromosome fusions, DNA damage responses, and cellular senescence, apoptosis, or malignancy.

(24) The enzyme complex telomerase extends telomeres and comprises two essential components: the telomerase reverse transcriptase (TERT), and an RNA component known as telomerase RNA component (TERC). Other components of the telomerase complex include the proteins TCAB1, Dyskerin, Gar1, Nhp2, Nop 10, and RHAU.

(25) Due to the importance of telomere length maintenance in preventing cellular senescence and apoptosis and resulting cellular dysfunction, genetic mutations of TERT and TERC are linked to fatal inherited diseases of inadequate telomere maintenance, including forms of idiopathic pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia. The effects of premature cellular senescence and apoptosis due to short telomeres in these diseases are devastating in themselves, and may be compounded by increased risk of cancer. Moreover, the shortening of telomeres in cells that are cultured in vitro results is also a major problem in the production of therapeutic cell populations, the creation of in vitro synthetic tissue and tumors and the in vitro creation of non-cancerous somatic cells lines for research and drug testing. Repeated passaging in vitro can lead to senescence and the lack of further expansion ability, and in the case of therapeutic cell populations, a decrease in clinically-relevant biological activity.

(26) Thus, there is a clear need in the art for compositions, kits and methods directed to elongating telomeres in order to rejuvenate cells. Existing approaches directed to increasing the expression of TERT and/or TERC in target cells has relied on the use of integrating viruses to obtain the desired increase in TERT and/or TERC expression. However, these approaches suffer from safety concerns, as the integrating viruses can result in potentially dangerous, permanent genome modifications. Moreover, the sustained overexpression of TERT and/or TERC, and concomitant increases in telomere length, have been linked to cancer cell immortalization, making the integrating virus approach dangerous in a clinical context.

(27) Without wishing to be bound by theory, the compositions, kits and methods of the present disclosure allow for the transient increase in TERT and/or TERC expression for a time period that is long enough to rejuvenate the target cells, but short enough to avoid deleterious and dangerous off-target effects. The use of non-integrating RNA molecules in the present disclosure allows for fine-tuning of the expression levels and stoichiometry of rejuvenating factors in a clinically safe manner.

(28) The compositions, kits and methods of the present disclosure can be used for a variety of different research and clinical applications, including, but not limited to, the production of therapeutic cell populations (e.g. CAR-T cell populations, mesenchymal stem/stromal cell populations), the production of in vitro tissue and organs for subsequent transplantation, research or drug testing, the production of genome-edited cell populations for therapeutic and research applications, the rejuvenation of senescent, aged and disease associated cell lines, etc.

(29) Various compositions, kits and methods of the present disclosure are described in full detail herein.

(30) Rejuvenating Compositions

(31) In some aspects, the present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(32) In some aspects, the present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(33) In some aspects, the at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) can be an mRNA molecule encoding at least a portion of TERT. In some aspects, the at least one first polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT.

(34) In some aspects, the at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide can be an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide. In some aspects, the at least one second polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide.

(35) Thus, the present disclosure provides a composition comprising: a) at least one first mRNA molecule encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(36) The present disclosure also provides a composition comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT; and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(37) The present disclosure also provides a composition comprising: a) at least one first mRNA molecule encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(38) The present disclosure also provides a composition comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT; and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(39) In some aspects, a DNA targeting polypeptide can comprise at least one Cas9 molecule, at least one Cas9 variant molecule, at least one Cas9 ortholog molecule or any combination thereof.

(40) In some aspects, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can be nuclease-deficient or nuclease-dead. As used herein, the term dCas9 is used in its broadest sense to refer to a Cas9 molecule, ortholog and/or variant that is nuclease-deficient or nuclease dead. In a non-limiting example, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can comprise at least one mutation, deletion or insertion which renders the Cas9 molecule, the Cas9 variant molecule or the Cas9 ortholog molecule nuclease-deficient or nuclease-dead.

(41) In some aspects, a Cas9 variant molecule can comprise eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D1135E), Cas9(QQR1), SaCas9 (KKH), Nme1 Cas9, Nme2Cas9, Nme3Cas9 or any combination thereof.

(42) In some aspects, a Cas9 ortholog molecule can comprise Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Cas (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

(43) In some aspects, a Cas9 ortholog molecule can comprise a chimeric variant of Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Cas (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.

(44) In some aspects, a DNA targeting polypeptide can comprise at least one TALE molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof.

(45) In some aspects, a DNA targeting polypeptide can comprise at least one transactivation molecule. In some aspects, a transactivation molecule is a molecule that binds to transcription factors and/or transcriptional co-regulators that are capable of driving transcription of a target gene.

(46) In some aspects, a transactivation molecule can comprise at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof.

(47) In some aspects, a DNA targeting polypeptide can be a DNA targeting ribonucleoprotein (RNP) complex. A DNA targeting ribonucleoprotein complex can comprise both at least one protein component and at least one nucleic acid component. The at least one protein component can comprise any of the protein components described herein, including, but not limited to, a transactivation molecule, a Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule, a TALE molecule, a zinc-finger molecule, a meganuclease molecule or any combination thereof. The at least one nucleic acid component can be a ribonucleic acid component. The at least one nucleic acid component can comprise any of the nucleic acid components described herein, including, but not limited to, a guide RNA molecule, a single guide RNA molecule, a single guide RNA-MS2 (sgRNA-MS2) molecule or any combination thereof.

(48) In some aspects, a DNA targeting polypeptide can further comprise at least one cell-penetrating peptide. A cell-penetrating peptide can comprise at least a portion of an HIV-derived TAT protein, polyarginine, any other cell-penetrating peptide known in the art or any combination thereof.

(49) In some aspects, a DNA targeting polypeptide can comprise at least one guide RNA. In some aspects, a transactivation molecule can comprise at least one single guide RNA-MS2 (sgRNA-MS2) molecule. In some aspects, a sgRNA-MS2 molecule can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

(50) In some aspects, a DNA targeting polypeptide can comprise a dCas9 molecule and a VPR molecule.

(51) In some aspects, a DNA targeting polypeptide can bind upstream of, 5 to, within, downstream of or 3 to the endogenous TERC gene, e.g. the endogenous human TERC gene.

(52) In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb upstream of the endogenous TERC gene, e.g. the endogenous human TERC gene.

(53) In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb 3 to the endogenous TERC gene, e.g. the endogenous human TERC gene.

(54) In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb downstream of the endogenous TERC gene, e.g. the endogenous human TERC gene.

(55) In some aspects, an at least one DNA targeting polypeptide can bind at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1000 kb, or at least 5000 kb or at least about 10,000 kb 5 to the endogenous TERC gene, e.g. the endogenous human TERC gene.

(56) In some aspects, an mRNA molecule of any composition of the present disclosure can be a modified mRNA molecule.

(57) In some aspects, a modified mRNA molecule can comprise at least one modified ribonucleoside base. A modified ribonucleoside base can comprise a pseudouridine () residue, a 5-methylcytidine (m.sup.5C) residue or any combination thereof.

(58) In some aspects, a modified mRNA molecule can comprise at least one modified nucleoside. A modified nucleoside can comprise 5-methylcytidine (m.sup.5C), 5-methyluridine (m.sup.5U), N6-methyladenosine (m.sup.6A), inosine and 2-0-methylated nucleosides, in addition to N7-methylguanosine (m.sup.7G), 2-thiouridine (s.sup.2U), pseudouridine (), 2-0-methyl-U, m.sup.1A (1-methyladenosine); m.sup.2A (2-methyladenosine); Am (2-0-methyladenosine); ms.sup.2m.sup.6A (2-methylthio-N.sup.6-methyladenosine); i.sup.6A (N.sup.6-isopentenyladenosine); ms.sup.2i6A (2-methylthio-N.sup.6isopentenyladenosine); io.sup.6A (N.sup.6-(cis-hydroxyisopentenyl)adenosine); ms.sup.2i.sup.6A (2-methylthio-N.sup.6-(cis-hydroxyisopentenyl)adenosine); g.sup.6A (N.sup.6-glycinylcarbamoyladenosine); t.sup.6A (N.sup.6-threonylcarbamoyladenosine); ms.sup.2t.sup.6A (2-methylthio-N.sup.6-threonyl carbamoyladenosine); m.sup.6t.sup.6A (N.sup.6-methyl-N.sup.6-threonylcarbamoyladenosine); hn.sup.6A(N.sup.6-hydroxynorvalylcarbamoyladenosine); ms.sup.2hn.sup.6A (2-methylthio-N.sup.6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2-0-ribosyladenosine(phosphate)); I (inosine); m.sup.1I (1-methylinosine); m.sup.1Im (1,2-0-dimethylinosine); m.sup.3C (3-methylcytidine); Cm (2-0-methylcytidine); s.sup.2C (2-thiocytidine); ac.sup.4C(N.sup.4-acetylcytidine); f.sup.5C (5-formylcytidine); m.sup.5 Cm (5,2-0-dimethylcytidine); ac.sup.4Cm (N.sup.4-acetyl-2-0-methylcytidine); k.sup.2C (lysidine); m.sup.1G (1-methylguanosine); m.sup.2G (N.sup.2-methylguanosine); m.sup.7G (7-methylguanosine); Gm (2-0-methylguanosine); m.sup.2.sub.2G (N.sup.2,N.sup.2-dimethylguanosine); m.sup.2Gm (N.sup.2,2-0-dimethylguanosine); m.sup.2.sub.2Gm (N.sup.2,N.sup.2,2-0-trimethylguanosine); Gr(p) (2-0-ribosylguanosine (phosphate)); yW (wybutosine); o.sub.2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ.sub.0 (7-cyano-7-deazaguanosine); preQ.sub.1 (7-aminomethyl-7-deazaguanosine); G.sup.+ (archaeosine); D (dihydrouridine); m.sup.5Um (5,2-0-dimethyluridine); s.sup.4U (4-thiouridine); m.sup.5s.sup.2U (5-methyl-2-thiouridine); s.sup.2Um (2-thio-2-0-methyluridine); acp.sup.3U (3-(3-amino-3-carboxypropyl)uridine); ho.sup.5U (5-hydroxyuridine); mo.sup.5U (5-methoxyuridine); cmo.sup.5U (uridine 5-oxyacetic acid); mcmo.sup.5U (uridine 5-oxyacetic acid methyl ester); chm.sup.5U (5-(carboxyhydroxymethyl)uridine)); mchm.sup.5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm.sup.5U (5-methoxycarbonylmethyluridine); mcm.sup.5Um (5-methoxycarbonylmethyl-2-0-methyluridine); mcm.sup.5s.sup.2U (5-methoxycarbonylmethyl-2-thiouridine); nm.sup.5s.sup.2U (5-aminomethyl-2-thiouridine); mnm.sup.5U (5-methylaminomethyluridine); mnm.sup.5s.sup.2U (5-methylaminomethyl-2-thiouridine); mnm.sup.5se.sup.2U (5-methylaminomethyl-2-selenouridine); ncm.sup.5U (5-carbamoylmethyluridine); ncm.sup.5Um (5-carbamoylmethyl-2-0-methyluridine); cmnm.sup.5U (5-carboxymethylaminomethyluridine); cmnm.sup.5Um (5-carboxymethylaminomethyl-2-0-methyluridine); cmnm.sup.5s.sup.2U (5-carboxymethylaminomethyl-2-thiouridine); m.sup.6.sub.2A (N.sup.6,N.sup.6-dimethyladenosine); Im (2-0-methylinosine); m.sup.4C(N.sup.4-methylcytidine); m.sup.4 Cm (N.sup.4,2-0-dimethylcytidine); hm.sup.5C (5-hydroxymethylcytidine); m.sup.3U (3-methyluridine); cm.sup.5U (5-carboxymethyluridine); m.sup.6Am (N.sup.6,2-0-dimethyladenosine); m.sup.6.sub.2Am (N.sup.6,N.sup.6,0-2-2 7 2 2 2 7 2 2trimethyladenosine); m.sup.2,7G (N.sup.2,7-dimethylguanosine); m.sup.2,2,7G (N.sup.2, N.sup.2,7-trimethylguanosine); m.sup.3Um (3,2-0-dimethyluridine); m.sup.5D (5-methyldihydrouridine); f.sup.5Cm (5-formyl-2-0-methylcytidine); m.sup.1Gm (1,2-0-dimethylguanosine); m.sup.1Am (1,2-0-dimethyladenosine); m.sup.5U (5-taurinomethyluridine); m.sup.5s.sup.2U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); ac.sup.6A (N.sup.6-acetyladenosine), or any combination thereof.

(59) In some aspects, an mRNA molecule can be chemically synthesized using methods standard in the art. In some aspects, an mRNA molecule can be chemically synthesized such that the mRNA molecule comprises at least one chemical modification. In some aspects, an mRNA molecule can be produced by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template, in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods can be performed such that the produced mRNA molecules comprise at least one chemical modification.

(60) In some aspects, a purified DNA targeting polypeptide can be produced using methods standard in the art, including, but not limited to, recombinant protein expression and purification in a bacterial, fungal, insect and/or mammalian system, ion-exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, and/or other standard protein production/purification methods known in the art.

(61) In some aspects, a purified DNA-targeting ribonucleoprotein (RNP) complex can be produced using methods standard in the art, including, but not limited to recombinant protein expression and purification in a bacterial, fungal, insect and/or mammalian system, in vitro RNA transcription, ion-exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, other standard protein production/purification methods known in the art, and/or other standard nucleic acid production/purification methods known in the art. In some aspects, a preassembled RNP complex that comprises both at least one protein component and at least one nucleic acid can be assembled in vivo (i.e. in a bacterial, fungal, insect and/or mammalian recombinant expression system) and co-purified. In some aspects, a RNP complex can be assembled in vitro after the individual purification of the at least one protein component and the at least one nucleic acid component.

(62) In some aspects, any of the compositions of the present disclosure can further comprise a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene. In some aspects, a plurality of gRNA molecules can comprise at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or about at least 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500 or at least about 1000 distinct species of gRNA molecules, wherein each species has a different nucleic acid sequence.

(63) In some aspects, any of the compositions of the present disclosure can further comprise at least one plasmid comprising at least one nucleic acid sequence encoding at least one species of gRNA operably linked to at least one promoter sufficient to drive expression of the at least one species gRNA. In some aspects, any of the compositions of the present disclosure can further comprise at least one plasmid comprising at least one nucleic acid sequence encoding least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or about at least 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500 or at least about 1000 distinct species of gRNA molecules operably linked to at least one promoter sufficient to drive expression of the gRNA species, wherein each species has a different nucleic acid sequence.

(64) In some aspects, a plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high fidelity scaffold gRNA molecules or any combination thereof.

(65) In some aspects, a plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules. In some aspects, a sgRNA molecule can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least one MS2 RNA aptamer. In some aspects, a sgRNA molecule can comprise at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

(66) In some aspects, a guide RNA molecule of any composition of the present disclosure can be a modified guide RNA (mod gRNA) molecule.

(67) In some aspects, a modified guide RNA can comprise at least one modified ribonucleoside base. A modified ribonucleoside base can comprise a pseudouridine () residue, a 5-methylcytidine (m.sup.5C) residue or any combination thereof.

(68) In some aspects, a modified guide RNA can comprise at least one modified nucleoside. A modified nucleoside can comprise 5-methylcytidine (m.sup.5C), 5-methyluridine (m.sup.5U), N6-methyladenosine (m.sup.6A), inosine and 2-0-methylated nucleosides, in addition to N7-methylguanosine (m.sup.7G), 2-thiouridine (s.sup.2U), pseudouridine (), 2-0-methyl-U, m.sup.1A (1-methyladenosine); m.sup.2A (2-methyladenosine); Am (2-0-methyladenosine); ms.sup.2m.sup.6A (2-methylthio-N.sup.6-methyladenosine); i.sup.6A (N.sup.6-isopentenyladenosine); ms.sup.2i6A (2-methylthio-N.sup.6isopentenyladenosine); io.sup.6A (N.sup.6-(cis-hydroxyisopentenyl)adenosine); ms.sup.2i.sup.6A (2-methylthio-N.sup.6-(cis-hydroxyisopentenyl)adenosine); g.sup.6A (N.sup.6-glycinylcarbamoyladenosine); t.sup.6A (N.sup.6-threonylcarbamoyladenosine); ms.sup.2t.sup.6A (2-methylthio-N.sup.6-threonyl carbamoyladenosine); m.sup.6t.sup.6A (N.sup.6-methyl-N.sup.6-threonylcarbamoyladenosine); hn.sup.6A(N.sup.6-hydroxynorvalylcarbamoyladenosine); ms.sup.2hn.sup.6A (2-methylthio-N.sup.6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2-0-ribosyladenosine(phosphate)); I (inosine); m.sup.1I (1-methylinosine); m.sup.1Im (1,2-0-dimethylinosine); m.sup.3C (3-methylcytidine); Cm (2-0-methylcytidine); s.sup.2C (2-thiocytidine); ac.sup.4C(N.sup.4-acetylcytidine); f.sup.5C (5-formylcytidine); m.sup.5Cm (5,2-0-dimethylcytidine); ac.sup.4Cm (N.sup.4-acetyl-2-0-methylcytidine); k.sup.2C (lysidine); m.sup.1G (1-methylguanosine); m.sup.2G (N.sup.2-methylguanosine); m.sup.7G (7-methylguanosine); Gm (2-0-methylguanosine); m.sup.2.sub.2G (N.sup.2,N.sup.2-dimethylguanosine); m.sup.2Gm (N.sup.2,2-0-dimethylguanosine); m.sup.2.sub.2Gm (N.sup.2,N.sup.2,2-0-trimethylguanosine); Gr(p) (2-0-ribosylguanosine (phosphate)); yW (wybutosine); o.sub.2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ.sub.0 (7-cyano-7-deazaguanosine); preQ.sub.1 (7-aminomethyl-7-deazaguanosine); G.sup.+ (archaeosine); D (dihydrouridine); m.sup.5Um (5,2-0-dimethyluridine); s.sup.4U (4-thiouridine); m.sup.5s.sup.2U (5-methyl-2-thiouridine); s.sup.2Um (2-thio-2-0-methyluridine); acp.sup.3U (3-(3-amino-3-carboxypropyl)uridine); ho.sup.5U (5-hydroxyuridine); mo.sup.5U (5-methoxyuridine); cmo.sup.5U (uridine 5-oxyacetic acid); mcmo.sup.5U (uridine 5-oxyacetic acid methyl ester); chm.sup.5U (5-(carboxyhydroxymethyl)uridine)); mchm.sup.5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm.sup.5U (5-methoxycarbonylmethyluridine); mcm.sup.5Um (5-methoxycarbonylmethyl-2-0-methyluridine); mcm.sup.5s.sup.2U (5-methoxycarbonylmethyl-2-thiouridine); nm.sup.5.sub.s.sup.2U (5-aminomethyl-2-thiouridine); mnm.sup.5U (5-methylaminomethyluridine); mnm.sup.5.sub.s.sup.2U (5-methylaminomethyl-2-thiouridine); mnm.sup.5se.sup.2U (5-methylaminomethyl-2-selenouridine); ncm.sup.5U (5-carbamoylmethyluridine); ncm.sup.5Um (5-carbamoylmethyl-2-0-methyluridine); cmnm.sup.5U (5-carboxymethylaminomethyluridine); cmnm.sup.5Um (5-carboxymethylaminomethyl-2-0-methyluridine); cmnm.sup.5.sub.s.sup.2U (5-carboxymethylaminomethyl-2-thiouridine); m.sup.6.sub.2A (N.sup.6,N.sup.6-dimethyladenosine); Im (2-0-methylinosine); m.sup.4C(N.sup.4-methylcytidine); m.sup.4 Cm (N.sup.4,2-0-dimethylcytidine); hm.sup.5C (5-hydroxymethylcytidine); m.sup.3U (3-methyluridine); cm.sup.5U (5-carboxymethyluridine); m.sup.6Am (N.sup.6,2-0-dimethyladenosine); m.sup.6.sub.2Am (N.sup.6,N.sup.6,0-2-2 7 2 2 2 7 2 2trimethyladenosine); m.sup.2,7G (N.sup.2,7-dimethylguanosine); m.sup.2,2,7G (N.sup.2,N.sup.2,7-trimethylguanosine); m.sup.3Um (3,2-0-dimethyluridine); m.sup.5D (5-methyldihydrouridine); f.sup.5Cm (5-formyl-2-0-methylcytidine); m.sup.1Gm (1,2-0-dimethylguanosine); m.sup.1Am (1,2-0-dimethyladenosine); m.sup.5U (5-taurinomethyluridine); m.sup.5.sub.s.sup.2U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); ac.sup.6A (N.sup.6-acetyladenosine), or any combination thereof.

(69) In some aspects, a guide RNA molecule can comprise any sequence recited in Table 1 or Table 2.

(70) TABLE-US-00001 TABLE1 GuideRNAsequences Seq SeqID Name Sequence NO 1015rev UGUUCAUAAAUUUACUGACA 1 1025forw AAAAAAAUCGUUACAAUUUA 2 1028forw AAAAUCGUUACAAUUUAUGG 3 1037rev UCUUGAUGAGGUAAAAAGAG 4 1038rev GUCUUGAUGAGGUAAAAAGA 5 1039rev UGUCUUGAUGAGGUAAAAAG 6 103rev AAUUUCUCUCCUUUGCAUAU 7 1049rev AGUAGUGCUGUGUCUUGAUG 8 1059rev AGGGGACCUACUUAGGUAAU 9 1066rev CAAUUCCAGGGGACCUACUU 10 106forw ACGGAGCGAGUCCCCGCGCG 11 1073forw UUUUAACCUAUUACCUAAGU 12 1077rev UAUCUGCUAGACAAUUCCAG 13 1078rev GUAUCUGCUAGACAAUUCCA 14 1079rev UGUAUCUGCUAGACAAUUCC 15 1081forw UAUUACCUAAGUAGGUCCCC 16 1098rev UCCCUUUUAUUAGGAAAGAA 17 1107rev GACUGAAUCUCCCUUUUAUU 18 1116forw CGCCUUUCUUUCCUAAUAAA 19 1117forw GCCUUUCUUUCCUAAUAAAA 20 1129rev CUACUACAUUAUUAAUCUUA 21 1139rev CCAGCAACAGUGGACUCUAG 22 1149rev GAGAACAUUACCAGCAACAG 23 114forw GCUAAAUAUCCAAUAUGCAA 24 1159forw CCUCUAGAGUCCACUGUUGC 25 1168rev GCCUCUCCUUGAGCAGAGGA 26 116rev GGUGCACGUCCCACAGCUCA 27 1172rev UCCAGCCUCUCCUUGAGCAG 28 1178forw CUGGUAAUGUUCUCUAAAUA 29 117rev GGGUGCACGUCCCACAGCUC 30 1182forw UUAAAGCCAUCCUCUGCUCA 31 1187forw GCCAUCCUCUGCUCAAGGAG 32 1191forw UCCUCUGCUCAAGGAGAGGC 33 1193rev UUCCACAAAACCAUGCUGAU 34 1197forw GCUCAAGGAGAGGCUGGAGA 35 1203forw AAAUAUUUUUCCUAUCAGCA 36 1207forw AGGCUGGAGAAGGCAUUCUA 37 1211forw UUCCUAUCAGCAUGGUUUUG 38 1213forw GAGAAGGCAUUCUAAGGAGA 39 1214forw AGAAGGCAUUCUAAGGAGAA 40 1215forw GAAGGCAUUCUAAGGAGAAG 41 1216forw AAGGCAUUCUAAGGAGAAGG 42 1220forw CAUUCUAAGGAGAAGGGGGC 43 1221forw AUUCUAAGGAGAAGGGGGCA 44 1221forw CAUGGUUUUGUGGAAAAGUA 45 1225forw UAAGGAGAAGGGGGCAGGGU 46 1232forw AAGGGGGCAGGGUAGGAACU 47 1234rev CAAGACUCUAGACAAGUUCU 48 1241rev GAAUCUUGUCUCGGCUCAGU 49 1242rev AGAAUCUUGUCUCGGCUCAG 50 1250rev ACUACAGCAGAAUCUUGUCU 51 1257forw AGAACUUGUCUAGAGUCUUG 52 126forw CGGCGCGAUUCCCUGAGCUG 53 127forw GGCGCGAUUCCCUGAGCUGU 54 1281rev CUUUGUGAAAAUAGAUUCCC 55 1281rev AGAUCACCUUGAGUAAACUG 56 1283forw CUGCUGUAGUCAGUGCUGCC 57 1284forw UGCUGUAGUCAGUGCUGCCU 58 1295forw AGUAAGCCUCAGUUUACUCA 59 1308rev GUUUUGAUCAUCACAUUUUU 60 1332forw AAAAUGUGAUGAUCAAAACU 61 1335forw UUCUUCUCUUUCUUUUGAGA 62 1341rev CCAGCUCUGGGUGACAGAGU 63 1342rev UCCAGCUCUGGGUGACAGAG 64 1353rev GGACACUGCACUCCAGCUCU 65 1354forw GAAUUAGUGUUCUGUGUCUU 66 1354rev GGGACACUGCACUCCAGCUC 67 1357rev GAAUUCACAGGAAGAUUUUA 68 1358rev GGAAUUCACAGGAAGAUUUU 69 1361forw CCCACUCUGUCACCCAGAGC 70 1369rev ACCUUAAAAAUGGAAUUCAC 71 1374rev GGUUGCAGUGAGCCAAGAUG 72 1375rev AGGUUGCAGUGAGCCAAGAU 73 1376rev GAGGUUGCAGUGAGCCAAGA 74 1379rev CACCUCGACUACCUUAAAAA 75 137rev GCAUGUGUGAGCCGAGUCCU 76 1382forw GGAGUGCAGUGUCCCCAUCU 77 1388forw UCCUGUGAAUUCCAUUUUUA 78 138rev UGCAUGUGUGAGCCGAGUCC 79 1395rev GCUAGAAACCGAGGAGGCAG 80 1397forw UUCCAUUUUUAAGGUAGUCG 81 1401rev AAAAUCGCUAGAAACCGAGG 82 1404rev UAUCCUCUGCAGACCAGACG 83 1404rev GAGAAAAUCGCUAGAAACCG 84 1407forw CACUGCAACCUCUGCCUCCU 85 140forw GAAAUUAAAGAUUUAAAAGC 86 140forw GAGCUGUGGGACGUGCACCC 87 1411forw UAGUCGAGGUGAACCGCGUC 88 1421forw GAACCGCGUCUGGUCUGCAG 89 1431rev UGUAAACCCAGCUACUUGGG 90 1433forw GUCUGCAGAGGAUAGAAAAA 91 1434rev GCCUGUAAACCCAGCUACUU 92 1435rev UGCCUGUAAACCCAGCUACU 93 1436rev AACUAACUUGAGGUAUCAGA 94 1437rev AAACUAACUUGAGGUAUCAG 95 1444forw CUCUCAGCCUCCCAAGUAGC 96 1445forw UCUCAGCCUCCCAAGUAGCU 97 1446rev UUAAAGGUGAAACUAACUUG 98 1453forw UCCCAAGUAGCUGGGUUUAC 99 145rev AUCAUAACAUAGUUUCCUUA 100 1462rev UUACUUCCGACCUUCUUUAA 101 1462rev AAAAAAUCAGCCGGGUAUGG 102 1465rev ACAAAAAAAUCAGCCGGGUA 103 146forw UGGGACGUGCACCCAGGACU 104 1470rev AAAAUACAAAAAAAUCAGCC 105 1471rev GAAAAUACAAAAAAAUCAGC 106 1472forw GUUAGUUUCACCUUUAAAGA 107 1472forw CAGGCACACACCACCAUACC 108 1476forw GUUUCACCUUUAAAGAAGGU 109 1495rev CCCUUCCGCACGUCCGGGAA 110 1500rev CGUUGCCCUUCCGCACGUCC 111 1501rev ACGUUGCCCUUCCGCACGUC 112 1502forw UAAAGACGCAAAGCCUUUCC 113 1502forw UUUUGUAUUUUCAGUAAAGU 114 1503forw UUUGUAUUUUCAGUAAAGUU 115 1507forw UAUUUUCAGUAAAGUUGGGC 116 150forw AUUUAAAAGCAGGAGCCAUA 117 1510forw CAAAGCCUUUCCCGGACGUG 118 1511forw UUCAGUAAAGUUGGGCAGGC 119 1514forw GCCUUUCCCGGACGUGCGGA 120 1514rev CACCUGAGGUCAGGAGUUCG 121 1515forw CCUUUCCCGGACGUGCGGAA 122 1523rev CGGGCGGAUCACCUGAGGUC 123 1524rev UCCAUUUCCGGCCAUGAGGA 124 1528rev AAGUUCCAUUUCCGGCCAUG 125 1528rev AGAAGCGGGCGGAUCACCUG 126 1532forw GGCCUCGAACUCCUGACCUC 127 1533forw AAGGGCAACGUCCUUCCUCA 128 1536rev GGAAAUUAAAGUUCCAUUUC 129 1537forw GCAACGUCCUUCCUCAUGGC 130 1539rev CUUUGGGAGGCAGAAGCGGG 131 1542rev GCACUUUGGGAGGCAGAAGC 132 1543forw UCCUUCCUCAUGGCCGGAAA 133 1543rev AGCACUUUGGGAGGCAGAAG 134 1552rev UGUAAUCCCAGCACUUUGGG 135 1555rev GCCUGUAAUCCCAGCACUUU 136 1556rev CGCCUGUAAUCCCAGCACUU 137 1557rev GCGGGCUGGUUGGGGGGAAC 138 1558rev GGCGGGCUGGUUGGGGGGAA 139 1563rev UCUCGGGCGGGCUGGUUGGG 140 1564rev CUCUCGGGCGGGCUGGUUGG 141 1565forw GCUUCUGCCUCCCAAAGUGC 142 1565rev UCUCUCGGGCGGGCUGGUUG 143 1566forw CUUCUGCCUCCCAAAGUGCU 144 1566rev CUCUCUCGGGCGGGCUGGUU 145 1567rev ACUCUCUCGGGCGGGCUGGU 146 1571rev AGUCACUCUCUCGGGCGGGC 147 1574forw UCCCAAAGUGCUGGGAUUAC 148 1575rev UGAGAGUCACUCUCUCGGGC 149 1576rev GUGAGAGUCACUCUCUCGGG 150 1579rev CUCGUGAGAGUCACUCUCUC 151 1580rev UCUCGUGAGAGUCACUCUCU 152 1583rev GGAUCUUAGUCCCCGCACGG 153 1586rev AAGGGAUCUUAGUCCCCGCA 154 1591forw UACAGGCGUGAGCCACCGUG 155 1592forw ACAGGCGUGAGCCACCGUGC 156 1593forw CAGGCGUGAGCCACCGUGCG 157 1604rev AUUGGCCAAGCUGACUCUCG 158 1604rev GAGUCCCCGCCCUUGCAAAA 159 1605rev GGAGUCCCCGCCCUUGCAAA 160 1614forw GGACUAAGAUCCCUUUUGCA 161 1615forw GACUAAGAUCCCUUUUGCAA 162 1618forw UAAGAUCCCUUUUGCAAGGG 163 1619forw GAGAGCCGCGAGAGUCAGCU 164 1619forw AAGAUCCCUUUUGCAAGGGC 165 1620forw AGAUCCCUUUUGCAAGGGCG 166 1622rev CGGCCGCCGACCGCACGGAU 167 1626rev AUGCACUUGUCUGUAGUUCA 168 1627rev GGGAGCGGCCGCCGACCGCA 169 1632forw GUCAGCUUGGCCAAUCCGUG 170 1636forw GCUUGGCCAAUCCGUGCGGU 171 1639forw UGGCCAAUCCGUGCGGUCGG 172 1642rev GAGUCGGCUUAUAAAGGGAG 173 1647rev CGGGCGAGUCGGCUUAUAAA 174 1648rev CCGGGCGAGUCGGCUUAUAA 175 1658rev CGGUGCGCUGCCGGGCGAGU 176 1665rev GAAGCAAAAGUACCACUAGA 177 1666rev CCGCAACCCGGUGCGCUGCC 178 1667rev UCCGCAACCCGGUGCGCUGC 179 1668forw CCUUUAUAAGCCGACUCGCC 180 1673forw UUUGUUCUUACUCCAUCUAG 181 1678rev CAGGCCCACCCUCCGCAACC 182 1679forw CGACUCGCCCGGCAGCGCAC 183 1680forw GACUCGCCCGGCAGCGCACC 184 1686forw CCCGGCAGCGCACCGGGUUG 185 1689forw GGCAGCGCACCGGGUUGCGG 186 1689rev CACCACAAAUGUUGUAAAUG 187 1690forw GCAGCGCACCGGGUUGCGGA 188 1693forw GCGCACCGGGUUGCGGAGGG 189 1694forw CGCACCGGGUUGCGGAGGGU 190 1697rev AAAUGGCCACCACCCCUCCC 191 1699forw CGGGUUGCGGAGGGUGGGCC 192 1700forw GGGUUGCGGAGGGUGGGCCU 193 1703forw UUGCGGAGGGUGGGCCUGGG 194 1704forw UGCGGAGGGUGGGCCUGGGA 195 1705forw GCGGAGGGUGGGCCUGGGAG 196 1707forw CUCCACAUUUACAACAUUUG 197 1708forw GAGGGUGGGCCUGGGAGGGG 198 170rev UCGGCGUUCCCCCCACCAAC 199 1710forw CACAUUUACAACAUUUGUGG 200 1711forw GGUGGGCCUGGGAGGGGUGG 201 1714rev AGUUAGGGUUAGACAAAAAA 202 1716forw UACAACAUUUGUGGUGGUGC 203 1717forw ACAACAUUUGUGGUGGUGCA 204 1720rev CUGUGGCCAUUCUUGCUUCA 205 1729rev GCCUACGCCCUUCUCAGUUA 206 1730rev CGCCUACGCCCUUCUCAGUU 207 1734forw GCAGGGCCGUGAAGCAAGAA 208 1737rev AGAAAAACAUUCCCAGUCUG 209 1741forw UUGUCUAACCCUAACUGAGA 210 1742forw UGUCUAACCCUAACUGAGAA 211 1745forw AAGCAAGAAUGGCCACAGAC 212 1746forw AGCAAGAAUGGCCACAGACU 213 1748forw ACCCUAACUGAGAAGGGCGU 214 1753rev GCGCGCGGGGAGCAAAAGCA 215 175forw CAUGCAGUUCGCUUUCCUGU 216 1766rev AGCGAGAAAAACAGCGCGCG 217 1767rev CAGCGAGAAAAACAGCGCGC 218 1768rev UCAGCGAGAAAAACAGCGCG 219 178forw GCAGUUCGCUUUCCUGUUGG 220 1798forw UUUUUCUCGCUGACUUUCAG 221 1799forw UUUUCUCGCUGACUUUCAGC 222 179forw CAGUUCGCUUUCCUGUUGGU 223 1802forw UCUCGCUGACUUUCAGCGGG 224 180forw AGUUCGCUUUCCUGUUGGUG 225 1810rev CGGUGGAAGGCGGCAGGCCG 226 1813forw UUCAGCGGGCGGAAAAGCCU 227 1816rev AAUGAACGGUGGAAGGCGGC 228 181forw UUAUGAUGAAUGUGAUAGUU 229 181forw GUUCGCUUUCCUGUUGGUGG 230 1820rev CUAGAAUGAACGGUGGAAGG 231 1823rev GCUCUAGAAUGAACGGUGGA 232 1827rev GUUUGCUCUAGAAUGAACGG 233 182forw UUCGCUUUCCUGUUGGUGGG 234 1830rev UUUGUUUGCUCUAGAAUGAA 235 1866forw AAACAAAAAAUGUCAGCUGC 236 1869rev GGUCCCCGGGAGGGGCGAAC 237 1870rev AGGUCCCCGGGAGGGGCGAA 238 1877rev CCGCCGCAGGUCCCCGGGAG 239 1878rev CCCGCCGCAGGUCCCCGGGA 240 1879rev ACCCGCCGCAGGUCCCCGGG 241 1882rev GCGACCCGCCGCAGGUCCCC 242 1883rev GGCGACCCGCCGCAGGUCCC 243 1884forw GCUGGCCCGUUCGCCCCUCC 244 1885forw CUGGCCCGUUCGCCCCUCCC 245 1886forw UGGCCCGUUCGCCCCUCCCG 246 1890rev CUGGGCAGGCGACCCGCCGC 247 1894forw UCGCCCCUCCCGGGGACCUG 248 1897forw CCCCUCCCGGGGACCUGCGG 249 1898forw CCCUCCCGGGGACCUGCGGC 250 189rev GGGUGACGGAUGCGCACGAU 251 1904rev GCGGGGUUCGGGGGCUGGGC 252 1908rev CCAGGCGGGGUUCGGGGGCU 253 1909rev UCCAGGCGGGGUUCGGGGGC 254 1913rev GGCCUCCAGGCGGGGUUCGG 255 1914rev CGGCCUCCAGGCGGGGUUCG 256 1915rev GCGGCCUCCAGGCGGGGUUC 257 1916rev CGCGGCCUCCAGGCGGGGUU 258 1921rev CCGACCGCGGCCUCCAGGCG 259 1922rev GCCGACCGCGGCCUCCAGGC 260 1923rev GGCCGACCGCGGCCUCCAGG 261 1926rev CCGGGCCGACCGCGGCCUCC 262 1928forw CCCAGCCCCCGAACCCCGCC 263 1931forw AGCCCCCGAACCCCGCCUGG 264 1934rev GAGAAGCCCCGGGCCGACCG 265 1937forw CGAACCCCGCCUGGAGGCCG 266 1941forw CCCCGCCUGGAGGCCGCGGU 267 1944rev GGUGCCUCCGGAGAAGCCCC 268 1945rev GGGUGCCUCCGGAGAAGCCC 269 1946forw CCUGGAGGCCGCGGUCGGCC 270 1947forw CUGGAGGCCGCGGUCGGCCC 271 1948forw UGGAGGCCGCGGUCGGCCCG 272 1956rev GCGGUGGCAGUGGGUGCCUC 273 1957forw CGGUCGGCCCGGGGCUUCUC 274 1960forw UCGGCCCGGGGCUUCUCCGG 275 1965rev CAACUCUUCGCGGUGGCAGU 276 1966rev CCAACUCUUCGCGGUGGCAG 277 1986forw CCACUGCCACCGCGAAGAGU 278 1987forw CACUGCCACCGCGAAGAGUU 279 199forw UUUGGAGAAUAAAUUGAAUG 280 1rev CAGAGCCCAACUCUUCGCGG 281 203forw GAGAAUAAAUUGAAUGAGGA 282 203rev CCAUUGCCGGCGAGGGGUGA 283 206rev AACUGAUCACCAAAUCUCCA 284 207rev UAACUGAUCACCAAAUCUCC 285 209forw AAAUUGAAUGAGGAAGGCCC 286 209rev AAGCCCCCAUUGCCGGCGAG 287 210rev CAAGCCCCCAUUGCCGGCGA 288 211rev ACAAGCCCCCAUUGCCGGCG 289 216rev GGUUCACAAGCCCCCAUUGC 290 217forw UGAGGAAGGCCCUGGAGAUU 291 217forw GCGCAUCCGUCACCCCUCGC 292 223forw CCGUCACCCCUCGCCGGCAA 293 224forw CGUCACCCCUCGCCGGCAAU 294 225forw GUCACCCCUCGCCGGCAAUG 295 226forw UCACCCCUCGCCGGCAAUGG 296 237rev GCCCAGUCAGUCAGGUUUGG 297 238rev GGCCCAGUCAGUCAGGUUUG 298 239rev UGGCCCAGUCAGUCAGGUUU 299 240rev CUGGCCCAGUCAGUCAGGUU 300 243rev AAGACUUGGCACUUUAUAUG 301 245rev GCACACUGGCCCAGUCAGUC 302 255forw AACCCCCAAACCUGACUGAC 303 256forw ACCCCCAAACCUGACUGACU 304 257rev AUAAUCUUGAGUACAAGACU 305 259rev CCUGCCAAUUUGCAGCACAC 306 275forw UGGGCCAGUGUGCUGCAAAU 307 279forw CCAGUGUGCUGCAAAUUGGC 308 287forw UACUCAAGAUUAUAAGCAAU 309 28rev CCUCGCCCCCGAGAGACCCG 310 290forw CAAAUUGGCAGGAGACGUGA 311 295rev UUCAUUUUGGCCGACUUUGG 312 298rev CCAUUCAUUUUGGCCGACUU 313 305forw CGUGAAGGCACCUCCAAAGU 314 308rev GGCUCACUGCCCAUUCAUUU 315 318forw CCAAAGUCGGCCAAAAUGAA 316 319forw CAAAGUCGGCCAAAAUGAAU 317 31forw GAGUUGGGCUCUGUCAGCCG 318 329rev GGAACGGCUCCAGGCAACCC 319 32forw AGUUGGGCUCUGUCAGCCGC 320 330forw AAAAUGAAUGGGCAGUGAGC 321 331forw AAAUGAAUGGGCAGUGAGCC 322 331rev UUUCCCCUUCAUAUCUAAGU 323 332forw AAUGAAUGGGCAGUGAGCCG 324 338rev ACCCACGCAGGAACGGCUCC 325 340forw GGCAGUGAGCCGGGGUUGCC 326 345rev CGGGAGAACCCACGCAGGAA 327 346forw UAGUGCCUACUUAGAUAUGA 328 347forw AGUGCCUACUUAGAUAUGAA 329 348forw GUGCCUACUUAGAUAUGAAG 330 350rev GAAGACGGGAGAACCCACGC 331 356forw UUAGAUAUGAAGGGGAAAGA 332 356forw UGCCUGGAGCCGUUCCUGCG 333 357forw UAGAUAUGAAGGGGAAAGAA 334 357forw GCCUGGAGCCGUUCCUGCGU 335 364rev GGCAACAAAAAGCGGAAGAC 336 365rev AGGCAACAAAAAGCGGAAGA 337 372forw AAGAAGGGUUUGAGAUAAUG 338 372rev CCAUAAAAGGCAACAAAAAG 339 373forw AGAAGGGUUUGAGAUAAUGU 340 385rev AGUUGUAAUACAACCAUAAA 341 388forw AAUGUGGGAUGCUAAGAGAA 342 391forw GUGGGAUGCUAAGAGAAUGG 343 392forw CCGCUUUUUGUUGCCUUUUA 344 40forw UCUGUCAGCCGCGGGUCUCU 345 413rev CUCAACAAAAUCUGCAGAGC 346 41forw CUGUCAGCCGCGGGUCUCUC 347 42forw UGUCAGCCGCGGGUCUCUCG 348 434forw CUGCUCUGCAGAUUUUGUUG 349 439forw UUUAGCAUCUACUCUAUGUA 350 43forw GUCAGCCGCGGGUCUCUCGG 351 448rev GACUGGUCGAGAUCUACCUU 352 449rev GGACUGGUCGAGAUCUACCU 353 452forw UGAGGUUUUUGCUUCUCCCA 354 465rev CCACACCCCGUUGAGGGGAC 355 470rev UUCUCCCACACCCCGUUGAG 356 471rev GUUCUCCCACACCCCGUUGA 357 472forw AGUGCAAUAGUGCUAAAAAC 358 472rev UGUUCUCCCACACCCCGUUG 359 478forw AUCUCGACCAGUCCCCUCAA 360 479forw UCUCGACCAGUCCCCUCAAC 361 480forw CUCGACCAGUCCCCUCAACG 362 485forw CCAGUCCCCUCAACGGGGUG 363 486forw CAGUCCCCUCAACGGGGUGU 364 488rev CCAGGUUGUAAAGUUUUUUA 365 48forw CCGCGGGUCUCUCGGGGGCG 366 49forw CGCGGGUCUCUCGGGGGCGA 367 4rev UGACAGAGCCCAACUCUUCG 368 506rev UUUCUUUCAUAGCAUCUGCC 369 508forw CCGUAAAAAACUUUACAACC 370 530forw GCAGAUGCUAUGAAAGAAAA 371 531forw CAGAUGCUAUGAAAGAAAAA 372 532forw AGAUGCUAUGAAAGAAAAAG 373 536forw GCUAUGAAAGAAAAAGGGGA 374 537forw CUAUGAAAGAAAAAGGGGAU 375 549forw AAGGGGAUGGGAGAGAGAGA 376 54forw GUCUCUCGGGGGCGAGGGCG 377 552forw GGGAUGGGAGAGAGAGAAGG 378 553forw GGAUGGGAGAGAGAGAAGGA 379 561forw UAGAAGAUCUAAAUGAACAU 380 563forw GAGAGAAGGAGGGAGAGAGA 381 568forw AAGGAGGGAGAGAGAUGGAG 382 569forw AGGAGGGAGAGAGAUGGAGA 383 574rev CAUAAACCGAUGACCAUUAA 384 57forw AACAAGCGCUAUGACUAGCA 385 581forw UGGAAAUUGUGUUCCUUUAA 386 588forw UGUGUUCCUUUAAUGGUCAU 387 597rev AAAAAGAAACUUCUAACCUC 388 598forw UUUACUUUUCUUUCAGAUCG 389 601forw UGGUCAUCGGUUUAUGCCAG 390 602rev CUCCGUGGAGUUGUCGCUGU 391 60forw CGGGGGCGAGGGCGAGGUUC 392 617rev AUUCAGUUAGAUAAACUCCG 393 620forw GACCGACAGCGACAACUCCA 394 632rev ACUGCUCAAGGUCAUCGCCA 395 635forw UUUUUUGAAAAAUUAGACCU 396 63rev UCCUCUUCCUGCGGCCUGAA 397 644rev GGGUUAUAUCCUACUGCUCA 398 655forw UGGCGAUGACCUUGAGCAGU 399 663rev CAGUUUUACAUAUAAAUGAC 400 664rev UUGGAACGCUAAGCUUGUGG 401 665rev AUUGGAACGCUAAGCUUGUG 402 666rev UAUUGGAACGCUAAGCUUGU 403 667rev UUAUUGGAACGCUAAGCUUG 404 683rev UAUGCCUAGUGUUCCGUUAU 405 68forw UGACUAGCAAGGUUAAGUGA 406 690forw CAAGCUUAGCGUUCCAAUAA 407 694forw UAUGUAAAACUGCACUAUAC 408 697rev CCGGCCGCGAAUUUUUAUAA 409 699forw CGUUCCAAUAACGGAACACU 410 69forw GGGCGAGGUUCAGGCCUUUC 411 713forw CUGGCCAUUAUAAAAAUUCG 412 714forw ACACUAGGCAUAAUGAAAGA 413 716rev CAGGUAUGAGCCACCGCACC 414 717forw CCAUUAUAAAAAUUCGCGGC 415 718forw CAUUAUAAAAAUUCGCGGCC 416 71rev ACUUUAAGCCUUUCAGUCCC 417 723forw UAAAAAUUCGCGGCCGGGUG 418 726forw AAAUUCGCGGCCGGGUGCGG 419 72rev UCGCUCCGUUCCUCUUCCUG 420 731rev AGGGUUGGGGGUGGGGGGUG 421 735rev UCCCAAAGUGCUGGGAUUAC 422 736rev CUGGGAGGGUUGGGGGUGGG 423 737rev GCUGGGAGGGUUGGGGGUGG 424 738rev GGCUGGGAGGGUUGGGGGUG 425 739rev CGGCUGGGAGGGUUGGGGGU 426 73forw AGCAAGGUUAAGUGAAGGCC 427 740rev CCGGCUGGGAGGGUUGGGGG 428 743rev CUUCGGCCUCCCAAAGUGCU 429 743rev CUGCCGGCUGGGAGGGUUGG 430 744rev GCUUCGGCCUCCCAAAGUGC 431 744rev ACUGCCGGCUGGGAGGGUUG 432 745rev GACUGCCGGCUGGGAGGGUU 433 746rev AGACUGCCGGCUGGGAGGGU 434 74forw GCAAGGUUAAGUGAAGGCCA 435 750rev UGGGAGACUGCCGGCUGGGA 436 751rev GUGGGAGACUGCCGGCUGGG 437 753forw UACCUGUAAUCCCAGCACUU 438 754forw ACCUGUAAUCCCAGCACUUU 439 754rev CUUGUGGGAGACUGCCGGCU 440 755rev UCUUGUGGGAGACUGCCGGC 441 757forw UGUAAUCCCAGCACUUUGGG 442 759rev CAAUUCUUGUGGGAGACUGC 443 760forw CCACCCCCAACCCUCCCAGC 444 760rev CUCAAGUGAUCCACCCGCUU 445 766forw AGCACUUUGGGAGGCCGAAG 446 767forw GCACUUUGGGAGGCCGAAGC 447 769rev AAAUCAGAGCCAAUUCUUGU 448 76forw GUUCAGGCCUUUCAGGCCGC 449 770forw CUUUGGGAGGCCGAAGCGGG 450 770rev GAAAUCAGAGCCAAUUCUUG 451 780forw CGGCAGUCUCCCACAAGAAU 452 783rev CAGGCUGGUCUCGAACGCCA 453 784rev CCAGGCUGGUCUCGAACGCC 454 786forw CGGGUGGAUCACUUGAGCCC 455 792rev CCAUUAGCUUAUUUUCUUAA 456 798rev UUUCACCAUGUUGCCCAGGC 457 802rev GGGGUUUCACCAUGUUGCCC 458 804forw CCUGGCGUUCGAGACCAGCC 459 805forw CUGGCGUUCGAGACCAGCCU 460 812forw CCUUUAAGAAAAUAAGCUAA 461 813forw CGAGACCAGCCUGGGCAACA 462 815rev UUUGUUUCUUUCAACCUAGU 463 816rev GUUUGUUUCUUUCAACCUAG 464 821forw AAAUAAGCUAAUGGCCCACU 465 821rev UGUGUUUUUAGUAGAGACGG 466 822rev UUGUGUUUUUAGUAGAGACG 467 823rev UUUGUGUUUUUAGUAGAGAC 468 824rev UUUUGUGUUUUUAGUAGAGA 469 82forw GCCUUUCAGGCCGCAGGAAG 470 839forw CUAGGUUGAAAGAAACAAAC 471 83forw AGUGAAGGCCAGGGACUGAA 472 842rev GUCGUGAUAAGUGGGCAGAA 473 850rev AUUACCUUGUCGUGAUAAGU 474 851rev AAUUACCUUGUCGUGAUAAG 475 853forw CUAAAAACACAAAAACUAGC 476 854forw UAAAAACACAAAAACUAGCU 477 859forw ACACAAAAACUAGCUGGGCG 478 862forw CAAAAACUAGCUGGGCGUGG 479 866forw AACUAGCUGGGCGUGGUGGC 480 866forw UCUGCCCACUUAUCACGACA 481 871rev UCCUGAGUAGCUGGGAUUAC 482 874rev UUGAAGGUAUGGAUUUGGGA 483 878rev GGAAUUGAAGGUAUGGAUUU 484 879rev UCUCAGCCUCCUGAGUAGCU 485 879rev AGGAAUUGAAGGUAUGGAUU 486 87forw UCAGGCCGCAGGAAGAGGAA 487 880rev GUCUCAGCCUCCUGAGUAGC 488 885rev AUCCUAAGGAAUUGAAGGUA 489 890forw GCCUGUAAUCCCAGCUACUC 490 890rev AGAUGAUCCUAAGGAAUUGA 491 893forw UGUAAUCCCAGCUACUCAGG 492 899rev ACUACCCCCAGAUGAUCCUA 493 903forw AUCCAUACCUUCAAUUCCUU 494 912forw UUCAAUUCCUUAGGAUCAUC 495 913forw UCAAUUCCUUAGGAUCAUCU 496 914forw CAAUUCCUUAGGAUCAUCUG 497 915forw AAUUCCUUAGGAUCAUCUGG 498 919rev CACUGCAACCUCUGCCUCCC 499 920rev UCACUGCAACCUCUGCCUCC 500 921forw ACACGAGAAUCGCUUGAACC 501 922forw CACGAGAAUCGCUUGAACCC 502 924rev CCCCUGGCUGCUCUCUCUCU 503 925forw GAGAAUCGCUUGAACCCGGG 504 931forw CGCUUGAACCCGGGAGGCAG 505 940rev GGCCUUUAUAUACACACCCC 506 942forw UGCCAAGAGAGAGAGCAGCC 507 943forw GCCAAGAGAGAGAGCAGCCA 508 944forw CCAAGAGAGAGAGCAGCCAG 509 944rev GGAGUCUAGUGGCGUGAUCU 510 955rev CCAGGCUGGAUGGAGUCUAG 511 958forw AGCCAGGGGUGUGUAUAUAA 512 961rev CAGGCUAUCACCCUAAAGGU 513 962rev UCAGGCUAUCACCCUAAAGG 514 965rev GCUCUUUCGCCCAGGCUGGA 515 965rev GAUUCAGGCUAUCACCCUAA 516 969rev UCUUGCUCUUUCGCCCAGGC 517 970forw GUAUAUAAAGGCCCACCUUU 518 971forw UAUAUAAAGGCCCACCUUUA 519 973rev GGAGUCUUGCUCUUUCGCCC 520 975forw CCACUAGACUCCAUCCAGCC 521 976forw CACUAGACUCCAUCCAGCCU 522 97rev AGGGAAUCGCGCCGCGCGCG 523 980rev ACUUUCAAUCAUCAGGAUUC 524 987rev ACUUCUGACUUUCAAUCAUC 525 98rev CAGGGAAUCGCGCCGCGCGC 526 994rev AACGAUUUUUUUUUUUGAGA 527 99rev UCAGGGAAUCGCGCCGCGCG 528

(71) TABLE-US-00002 TABLE2 GuideRNAsequences Seq SeqID Name Sequence NO 1420forw GUGAACCGCGUCUGGUCUGCA 529 1591rev GCUGACUCUCGCGGCUCUCGU 530 634forw AACUCCACGGAGUUUAUCUAA 531 640forw ACGGAGUUUAUCUAACUGAAU 532 1688forw CCGGCAGCGCACCGGGUUGCG 533 716forw GGCCAUUAUAAAAAUUCGCGG 534 1608forw AGUGACUCUCACGAGAGCCGC 535 1678forw GCCGACUCGCCCGGCAGCGCA 536 1728rev GGCGCCUACGCCCUUCUCAGU 537 620forw GGACCGACAGCGACAACUCCA 538 1239rev CAGAAUCUUGUCUCGGCUCAG 539 1229rev UCUCGGCUCAGUGGGAUGCGU 540 1584forw CCCCCCAACCAGCCCGCCCGA 541 481rev GGUUGUAAAGUUUUUUACGGA 542 1625rev AAGGGAGCGGCCGCCGACCGC 543 1896forw CGCCCCUCCCGGGGACCUGCG 544 1661rev CGCAACCCGGUGCGCUGCCGG 545 383forw UGAGAUAAUGUGGGAUGCUAA 546 472forw AAGUGCAAUAGUGCUAAAAAC 547 614rev UAUUCAGUUAGAUAAACUCCG 548 1376rev UCACCUCGACUACCUUAAAAA 549 166forw CCAUAAGGAAACUAUGUUAUG 550 409rev AUAGAGUAGAUGCUAAAUGCU 551 1170rev UCUCCAGCCUCUCCUUGAGCA 552 1122rev CUACAUUAUUAAUCUUAAGGA 553 1372forw CUUAGGCCCUAAAAUCUUCCU 554 268rev AAAUUCCUAUUGCUUAUAAUC 555 1836rev GCUGACAUUUUUUGUUUGCUC 556 183forw UAUGAUGAAUGUGAUAGUUUG 557 1260rev CCCAGGCAGCACUGACUACAG 558 1034rev GUCUUGAUGAGGUAAAAAGAG 559 1027forw AAAAAAUCGUUACAAUUUAUG 560 287forw GUACUCAAGAUUAUAAGCAAU 561 101rev UUAAUUUCUCUCCUUUGCAUA 562 1201rev CCUACCCUGCCCCCUUCUCCU 563 1284forw CUGCUGUAGUCAGUGCUGCCU 564 429rev CACUUAGCACAGUACCUUACA 565 355forw ACUUAGAUAUGAAGGGGAAAG 566 661rev UGCAGUUUUACAUAUAAAUGA 567 1703forw GUUGCGGAGGGUGGGCCUGGG 568 1332forw AAAAAUGUGAUGAUCAAAACU 569 1219forw GGCAUUCUAAGGAGAAGGGGG 570 372forw AAAGAAGGGUUUGAGAUAAUG 571 192forw UGUGAUAGUUUGGAGAAUAAA 572 531forw GCAGAUGCUAUGAAAGAAAAA 573 708rev UAUGAGCCACCGCACCCGGCC 574 741rev CGCUUCGGCCUCCCAAAGUGC 575 765forw CCAGCACUUUGGGAGGCCGAA 576 769forw CACUUUGGGAGGCCGAAGCGG 577 820rev UUUUGUGUUUUUAGUAGAGAC 578 877rev UGUCUCAGCCUCCUGAGUAGC 579 886rev CGAUUCUCGUGUCUCAGCCUC 580 905forw CUACUCAGGAGGCUGAGACAC 581 918rev GCUCACUGCAACCUCUGCCUC 582 962rev UGCUCUUUCGCCCAGGCUGGA 583 967rev AGUCUUGCUCUUUCGCCCAGG 584 991rev UAACGAUUUUUUUUUUUGAGA 585 94rev GCCCAACUCUUCGCGGUGGCA 586 302rev CCCCAUUGCCGGCGAGGGGUG 587 997rev AACUACCCCCAGAUGAUCCUA 588 237rev ACUGCAUGUGUGAGCCGAGUC 589 309rev CACAAGCCCCCAUUGCCGGCG 590 579forw GAUCUCGACCAGUCCCCUCAA 591 110forw GAGGCACCCACUGCCACCGCG 592 1082forw GCCCACCUUUAGGGUGAUAGC 593 456forw GUUGCCUGGAGCCGUUCCUGC 594 1391rev AAAAAGCGAUCUUAGAUCACC 595 414forw GCACCUCCAAAGUCGGCCAAA 596 1182forw CUAUUACCUAAGUAGGUCCCC 597 192forw GGCCGCAGGAAGAGGAACGGA 598 875forw UCCCAGCCGGCAGUCUCCCAC 599 1830forw GGUGGUGCAGGGCCGUGAAGC 600 214rev GGGUGCACGUCCCACAGCUCA 601 1724rev CAUGCACUUGUCUGUAGUUCA 602 1070forw GUGUAUAUAAAGGCCCACCUU 603 337rev CUGGCCCAGUCAGUCAGGUUU 604 850rev UUGUGGGAGACUGCCGGCUGG 605 984rev UGAUCCUAAGGAAUUGAAGGU 606 1086rev UGACUUCUGACUUUCAAUCAU 607 843rev AGACUGCCGGCUGGGAGGGUU 608 1014forw UUCAAUUCCUUAGGAUCAUCU 609 1348forw UGAUUUUGCCAAGAACUUGUC 610 1703rev AGGAGUCCCCGCCCUUGCAAA 611 244rev AAAGCGAACUGCAUGUGUGAG 612 945rev UACCUUGUCGUGAUAAGUGGG 613 1847forw AAGCAAGAAUGGCCACAGACU 614 1043forw UUGCCAAGAGAGAGAGCAGCC 615 50rev GGGCCGACCGCGGCCUCCAGG 616 131forw AAGAGUUGGGCUCUGUCAGCC 617 1217forw AGAUACAUUUCUUAGCACUAU 618 1763rev AGAAGCAAAAGUACCACUAGA 619 431forw CAAAAUGAAUGGGCAGUGAGC 620 1322forw GCAUGGUUUUGUGGAAAAGUA 621 972rev AUUGAAGGUAUGGAUUUGGGA 622 629forw UGAGAGAUCAUUUAACAUUUA 623 1003forw AAAUCCAUACCUUCAAUUCCU 624 1244forw UAAGAAAUGUAAAAAAACCUC 625 836rev CGGCUGGGAGGGUUGGGGGUG 626 1462forw UCCCACUCUGUCACCCAGAGC 627 764rev UUAUUGGAACGCUAAGCUUGU 628 829rev GAGGGUUGGGGGUGGGGGGUG 629 1445rev CUGCACUCCAGCUCUGGGUGA 630 755forw CUUGGCGAUGACCUUGAGCAG 631 1436forw UUUCUUCUCUUUCUUUUGAGA 632 1453rev UGGGGACACUGCACUCCAGCU 633 1544forw UUCUCUCAGCCUCCCAAGUAG 634 1570rev CUGAAAAUACAAAAAAAUCAG 635 1621rev GCGGGCGGAUCACCUGAGGUC 636 1638rev CACUUUGGGAGGCAGAAGCGG 637 1666forw CGCUUCUGCCUCCCAAAGUGC 638

(72) In some aspects, a guide RNA molecule can comprise a part of any sequence recited in Table 3 or Table 4. In some aspects, a guide RNA molecule can comprise the first about 20 nucleotides of any sequence recited in Table 3. In some aspects, a guide RNA molecule can comprise the first about 21 nucleotides of any sequence recited in Table 4.

(73) TABLE-US-00003 TABLE3 GuideRNAsequences Seq SeqID Name Sequence NO 1015rev UGUUCAUAAAUUUACUGACAUGG 639 1025forw AAAAAAAUCGUUACAAUUUAUGG 640 1028forw AAAAUCGUUACAAUUUAUGGUGG 641 1037rev UCUUGAUGAGGUAAAAAGAGGGG 642 1038rev GUCUUGAUGAGGUAAAAAGAGGG 643 1039rev UGUCUUGAUGAGGUAAAAAGAGG 644 103rev AAUUUCUCUCCUUUGCAUAUUGG 645 1049rev AGUAGUGCUGUGUCUUGAUGAGG 646 1059rev AGGGGACCUACUUAGGUAAUAGG 647 1066rev CAAUUCCAGGGGACCUACUUAGG 648 106forw ACGGAGCGAGUCCCCGCGCGCGG 649 1073forw UUUUAACCUAUUACCUAAGUAGG 650 1077rev UAUCUGCUAGACAAUUCCAGGGG 651 1078rev GUAUCUGCUAGACAAUUCCAGGG 652 1079rev UGUAUCUGCUAGACAAUUCCAGG 653 1081forw UAUUACCUAAGUAGGUCCCCUGG 654 1098rev UCCCUUUUAUUAGGAAAGAAAGG 655 1107rev GACUGAAUCUCCCUUUUAUUAGG 656 1116forw CGCCUUUCUUUCCUAAUAAAAGG 657 1117forw GCCUUUCUUUCCUAAUAAAAGGG 658 1129rev CUACUACAUUAUUAAUCUUAAGG 659 1139rev CCAGCAACAGUGGACUCUAGAGG 660 1149rev GAGAACAUUACCAGCAACAGUGG 661 114forw GCUAAAUAUCCAAUAUGCAAAGG 662 1159forw CCUCUAGAGUCCACUGUUGCUGG 663 1168rev GCCUCUCCUUGAGCAGAGGAUGG 664 116rev GGUGCACGUCCCACAGCUCAGGG 665 1172rev UCCAGCCUCUCCUUGAGCAGAGG 666 1178forw CUGGUAAUGUUCUCUAAAUAAGG 667 117rev GGGUGCACGUCCCACAGCUCAGG 668 1182forw UUAAAGCCAUCCUCUGCUCAAGG 669 1187forw GCCAUCCUCUGCUCAAGGAGAGG 670 1191forw UCCUCUGCUCAAGGAGAGGCUGG 671 1193rev UUCCACAAAACCAUGCUGAUAGG 672 1197forw GCUCAAGGAGAGGCUGGAGAAGG 673 1203forw AAAUAUUUUUCCUAUCAGCAUGG 674 1207forw AGGCUGGAGAAGGCAUUCUAAGG 675 1211forw UUCCUAUCAGCAUGGUUUUGUGG 676 1213forw GAGAAGGCAUUCUAAGGAGAAGG 677 1214forw AGAAGGCAUUCUAAGGAGAAGGG 678 1215forw GAAGGCAUUCUAAGGAGAAGGGG 679 1216forw AAGGCAUUCUAAGGAGAAGGGGG 680 1220forw CAUUCUAAGGAGAAGGGGGCAGG 681 1221forw AUUCUAAGGAGAAGGGGGCAGGG 682 1221forw CAUGGUUUUGUGGAAAAGUAAGG 683 1225forw UAAGGAGAAGGGGGCAGGGUAGG 684 1232forw AAGGGGGCAGGGUAGGAACUCGG 685 1234rev CAAGACUCUAGACAAGUUCUUGG 686 1241rev GAAUCUUGUCUCGGCUCAGUGGG 687 1242rev AGAAUCUUGUCUCGGCUCAGUGG 688 1250rev ACUACAGCAGAAUCUUGUCUCGG 689 1257forw AGAACUUGUCUAGAGUCUUGAGG 690 126forw CGGCGCGAUUCCCUGAGCUGUGG 691 127forw GGCGCGAUUCCCUGAGCUGUGGG 692 1281rev CUUUGUGAAAAUAGAUUCCCAGG 693 1281rev AGAUCACCUUGAGUAAACUGAGG 694 1283forw CUGCUGUAGUCAGUGCUGCCUGG 695 1284forw UGCUGUAGUCAGUGCUGCCUGGG 696 1295forw AGUAAGCCUCAGUUUACUCAAGG 697 1308rev GUUUUGAUCAUCACAUUUUUUGG 698 1332forw AAAAUGUGAUGAUCAAAACUAGG 699 1335forw UUCUUCUCUUUCUUUUGAGACGG 700 1341rev CCAGCUCUGGGUGACAGAGUGGG 701 1342rev UCCAGCUCUGGGUGACAGAGUGG 702 1353rev GGACACUGCACUCCAGCUCUGGG 703 1354forw GAAUUAGUGUUCUGUGUCUUAGG 704 1354rev GGGACACUGCACUCCAGCUCUGG 705 1357rev GAAUUCACAGGAAGAUUUUAGGG 706 1358rev GGAAUUCACAGGAAGAUUUUAGG 707 1361forw CCCACUCUGUCACCCAGAGCUGG 708 1369rev ACCUUAAAAAUGGAAUUCACAGG 709 1374rev GGUUGCAGUGAGCCAAGAUGGGG 710 1375rev AGGUUGCAGUGAGCCAAGAUGGG 711 1376rev GAGGUUGCAGUGAGCCAAGAUGG 712 1379rev CACCUCGACUACCUUAAAAAUGG 713 137rev GCAUGUGUGAGCCGAGUCCUGGG 714 1382forw GGAGUGCAGUGUCCCCAUCUUGG 715 1388forw UCCUGUGAAUUCCAUUUUUAAGG 716 138rev UGCAUGUGUGAGCCGAGUCCUGG 717 1395rev GCUAGAAACCGAGGAGGCAGAGG 718 1397forw UUCCAUUUUUAAGGUAGUCGAGG 719 1401rev AAAAUCGCUAGAAACCGAGGAGG 720 1404rev UAUCCUCUGCAGACCAGACGCGG 721 1404rev GAGAAAAUCGCUAGAAACCGAGG 722 1407forw CACUGCAACCUCUGCCUCCUCGG 723 140forw GAAAUUAAAGAUUUAAAAGCAGG 724 140forw GAGCUGUGGGACGUGCACCCAGG 725 1411forw UAGUCGAGGUGAACCGCGUCUGG 726 1421forw GAACCGCGUCUGGUCUGCAGAGG 727 1431rev UGUAAACCCAGCUACUUGGGAGG 728 1433forw GUCUGCAGAGGAUAGAAAAAAGG 729 1434rev GCCUGUAAACCCAGCUACUUGGG 730 1435rev UGCCUGUAAACCCAGCUACUUGG 731 1436rev AACUAACUUGAGGUAUCAGAGGG 732 1437rev AAACUAACUUGAGGUAUCAGAGG 733 1444forw CUCUCAGCCUCCCAAGUAGCUGG 734 1445forw UCUCAGCCUCCCAAGUAGCUGGG 735 1446rev UUAAAGGUGAAACUAACUUGAGG 736 1453forw UCCCAAGUAGCUGGGUUUACAGG 737 145rev AUCAUAACAUAGUUUCCUUAUGG 738 1462rev UUACUUCCGACCUUCUUUAAAGG 739 1462rev AAAAAAUCAGCCGGGUAUGGUGG 740 1465rev ACAAAAAAAUCAGCCGGGUAUGG 741 146forw UGGGACGUGCACCCAGGACUCGG 742 1470rev AAAAUACAAAAAAAUCAGCCGGG 743 1471rev GAAAAUACAAAAAAAUCAGCCGG 744 1472forw GUUAGUUUCACCUUUAAAGAAGG 745 1472forw CAGGCACACACCACCAUACCCGG 746 1476forw GUUUCACCUUUAAAGAAGGUCGG 747 1495rev CCCUUCCGCACGUCCGGGAAAGG 748 1500rev CGUUGCCCUUCCGCACGUCCGGG 749 1501rev ACGUUGCCCUUCCGCACGUCCGG 750 1502forw UAAAGACGCAAAGCCUUUCCCGG 751 1502forw UUUUGUAUUUUCAGUAAAGUUGG 752 1503forw UUUGUAUUUUCAGUAAAGUUGGG 753 1507forw UAUUUUCAGUAAAGUUGGGCAGG 754 150forw AUUUAAAAGCAGGAGCCAUAAGG 755 1510forw CAAAGCCUUUCCCGGACGUGCGG 756 1511forw UUCAGUAAAGUUGGGCAGGCUGG 757 1514forw GCCUUUCCCGGACGUGCGGAAGG 758 1514rev CACCUGAGGUCAGGAGUUCGAGG 759 1515forw CCUUUCCCGGACGUGCGGAAGGG 760 1523rev CGGGCGGAUCACCUGAGGUCAGG 761 1524rev UCCAUUUCCGGCCAUGAGGAAGG 762 1528rev AAGUUCCAUUUCCGGCCAUGAGG 763 1528rev AGAAGCGGGCGGAUCACCUGAGG 764 1532forw GGCCUCGAACUCCUGACCUCAGG 765 1533forw AAGGGCAACGUCCUUCCUCAUGG 766 1536rev GGAAAUUAAAGUUCCAUUUCCGG 767 1537forw GCAACGUCCUUCCUCAUGGCCGG 768 1539rev CUUUGGGAGGCAGAAGCGGGCGG 769 1542rev GCACUUUGGGAGGCAGAAGCGGG 770 1543forw UCCUUCCUCAUGGCCGGAAAUGG 771 1543rev AGCACUUUGGGAGGCAGAAGCGG 772 1552rev UGUAAUCCCAGCACUUUGGGAGG 773 1555rev GCCUGUAAUCCCAGCACUUUGGG 774 1556rev CGCCUGUAAUCCCAGCACUUUGG 775 1557rev GCGGGCUGGUUGGGGGGAACGGG 776 1558rev GGCGGGCUGGUUGGGGGGAACGG 777 1563rev UCUCGGGCGGGCUGGUUGGGGGG 778 1564rev CUCUCGGGCGGGCUGGUUGGGGG 779 1565forw GCUUCUGCCUCCCAAAGUGCUGG 780 1565rev UCUCUCGGGCGGGCUGGUUGGGG 781 1566forw CUUCUGCCUCCCAAAGUGCUGGG 782 1566rev CUCUCUCGGGCGGGCUGGUUGGG 783 1567rev ACUCUCUCGGGCGGGCUGGUUGG 784 1571rev AGUCACUCUCUCGGGCGGGCUGG 785 1574forw UCCCAAAGUGCUGGGAUUACAGG 786 1575rev UGAGAGUCACUCUCUCGGGCGGG 787 1576rev GUGAGAGUCACUCUCUCGGGCGG 788 1579rev CUCGUGAGAGUCACUCUCUCGGG 789 1580rev UCUCGUGAGAGUCACUCUCUCGG 790 1583rev GGAUCUUAGUCCCCGCACGGUGG 791 1586rev AAGGGAUCUUAGUCCCCGCACGG 792 1591forw UACAGGCGUGAGCCACCGUGCGG 793 1592forw ACAGGCGUGAGCCACCGUGCGGG 794 1593forw CAGGCGUGAGCCACCGUGCGGGG 795 1604rev AUUGGCCAAGCUGACUCUCGCGG 796 1604rev GAGUCCCCGCCCUUGCAAAAGGG 797 1605rev GGAGUCCCCGCCCUUGCAAAAGG 798 1614forw GGACUAAGAUCCCUUUUGCAAGG 799 1615forw GACUAAGAUCCCUUUUGCAAGGG 800 1618forw UAAGAUCCCUUUUGCAAGGGCGG 801 1619forw GAGAGCCGCGAGAGUCAGCUUGG 802 1619forw AAGAUCCCUUUUGCAAGGGCGGG 803 1620forw AGAUCCCUUUUGCAAGGGCGGGG 804 1622rev CGGCCGCCGACCGCACGGAUUGG 805 1626rev AUGCACUUGUCUGUAGUUCAAGG 806 1627rev GGGAGCGGCCGCCGACCGCACGG 807 1632forw GUCAGCUUGGCCAAUCCGUGCGG 808 1636forw GCUUGGCCAAUCCGUGCGGUCGG 809 1639forw UGGCCAAUCCGUGCGGUCGGCGG 810 1642rev GAGUCGGCUUAUAAAGGGAGCGG 811 1647rev CGGGCGAGUCGGCUUAUAAAGGG 812 1648rev CCGGGCGAGUCGGCUUAUAAAGG 813 1658rev CGGUGCGCUGCCGGGCGAGUCGG 814 1665rev GAAGCAAAAGUACCACUAGAUGG 815 1666rev CCGCAACCCGGUGCGCUGCCGGG 816 1667rev UCCGCAACCCGGUGCGCUGCCGG 817 1668forw CCUUUAUAAGCCGACUCGCCCGG 818 1673forw UUUGUUCUUACUCCAUCUAGUGG 819 1678rev CAGGCCCACCCUCCGCAACCCGG 820 1679forw CGACUCGCCCGGCAGCGCACCGG 821 1680forw GACUCGCCCGGCAGCGCACCGGG 822 1686forw CCCGGCAGCGCACCGGGUUGCGG 823 1689forw GGCAGCGCACCGGGUUGCGGAGG 824 1689rev CACCACAAAUGUUGUAAAUGUGG 825 1690forw GCAGCGCACCGGGUUGCGGAGGG 826 1693forw GCGCACCGGGUUGCGGAGGGUGG 827 1694forw CGCACCGGGUUGCGGAGGGUGGG 828 1697rev AAAUGGCCACCACCCCUCCCAGG 829 1699forw CGGGUUGCGGAGGGUGGGCCUGG 830 1700forw GGGUUGCGGAGGGUGGGCCUGGG 831 1703forw UUGCGGAGGGUGGGCCUGGGAGG 832 1704forw UGCGGAGGGUGGGCCUGGGAGGG 833 1705forw GCGGAGGGUGGGCCUGGGAGGGG 834 1707forw CUCCACAUUUACAACAUUUGUGG 835 1708forw GAGGGUGGGCCUGGGAGGGGUGG 836 170rev UCGGCGUUCCCCCCACCAACAGG 837 1710forw CACAUUUACAACAUUUGUGGUGG 838 1711forw GGUGGGCCUGGGAGGGGUGGUGG 839 1714rev AGUUAGGGUUAGACAAAAAAUGG 840 1716forw UACAACAUUUGUGGUGGUGCAGG 841 1717forw ACAACAUUUGUGGUGGUGCAGGG 842 1720rev CUGUGGCCAUUCUUGCUUCACGG 843 1729rev GCCUACGCCCUUCUCAGUUAGGG 844 1730rev CGCCUACGCCCUUCUCAGUUAGG 845 1734forw GCAGGGCCGUGAAGCAAGAAUGG 846 1737rev AGAAAAACAUUCCCAGUCUGUGG 847 1741forw UUGUCUAACCCUAACUGAGAAGG 848 1742forw UGUCUAACCCUAACUGAGAAGGG 849 1745forw AAGCAAGAAUGGCCACAGACUGG 850 1746forw AGCAAGAAUGGCCACAGACUGGG 851 1748forw ACCCUAACUGAGAAGGGCGUAGG 852 1753rev GCGCGCGGGGAGCAAAAGCACGG 853 175forw CAUGCAGUUCGCUUUCCUGUUGG 854 1766rev AGCGAGAAAAACAGCGCGCGGGG 855 1767rev CAGCGAGAAAAACAGCGCGCGGG 856 1768rev UCAGCGAGAAAAACAGCGCGCGG 857 178forw GCAGUUCGCUUUCCUGUUGGUGG 858 1798forw UUUUUCUCGCUGACUUUCAGCGG 859 1799forw UUUUCUCGCUGACUUUCAGCGGG 860 179forw CAGUUCGCUUUCCUGUUGGUGGG 861 1802forw UCUCGCUGACUUUCAGCGGGCGG 862 180forw AGUUCGCUUUCCUGUUGGUGGGG 863 1810rev CGGUGGAAGGCGGCAGGCCGAGG 864 1813forw UUCAGCGGGCGGAAAAGCCUCGG 865 1816rev AAUGAACGGUGGAAGGCGGCAGG 866 181forw UUAUGAUGAAUGUGAUAGUUUGG 867 181forw GUUCGCUUUCCUGUUGGUGGGGG 868 1820rev CUAGAAUGAACGGUGGAAGGCGG 869 1823rev GCUCUAGAAUGAACGGUGGAAGG 870 1827rev GUUUGCUCUAGAAUGAACGGUGG 871 182forw UUCGCUUUCCUGUUGGUGGGGGG 872 1830rev UUUGUUUGCUCUAGAAUGAACGG 873 1866forw AAACAAAAAAUGUCAGCUGCUGG 874 1869rev GGUCCCCGGGAGGGGCGAACGGG 875 1870rev AGGUCCCCGGGAGGGGCGAACGG 876 1877rev CCGCCGCAGGUCCCCGGGAGGGG 877 1878rev CCCGCCGCAGGUCCCCGGGAGGG 878 1879rev ACCCGCCGCAGGUCCCCGGGAGG 879 1882rev GCGACCCGCCGCAGGUCCCCGGG 880 1883rev GGCGACCCGCCGCAGGUCCCCGG 881 1884forw GCUGGCCCGUUCGCCCCUCCCGG 882 1885forw CUGGCCCGUUCGCCCCUCCCGGG 883 1886forw UGGCCCGUUCGCCCCUCCCGGGG 884 1890rev CUGGGCAGGCGACCCGCCGCAGG 885 1894forw UCGCCCCUCCCGGGGACCUGCGG 886 1897forw CCCCUCCCGGGGACCUGCGGCGG 887 1898forw CCCUCCCGGGGACCUGCGGCGGG 888 189rev GGGUGACGGAUGCGCACGAUCGG 889 1904rev GCGGGGUUCGGGGGCUGGGCAGG 890 1908rev CCAGGCGGGGUUCGGGGGCUGGG 891 1909rev UCCAGGCGGGGUUCGGGGGCUGG 892 1913rev GGCCUCCAGGCGGGGUUCGGGGG 893 1914rev CGGCCUCCAGGCGGGGUUCGGGG 894 1915rev GCGGCCUCCAGGCGGGGUUCGGG 895 1916rev CGCGGCCUCCAGGCGGGGUUCGG 896 1921rev CCGACCGCGGCCUCCAGGCGGGG 897 1922rev GCCGACCGCGGCCUCCAGGCGGG 898 1923rev GGCCGACCGCGGCCUCCAGGCGG 899 1926rev CCGGGCCGACCGCGGCCUCCAGG 900 1928forw CCCAGCCCCCGAACCCCGCCUGG 901 1931forw AGCCCCCGAACCCCGCCUGGAGG 902 1934rev GAGAAGCCCCGGGCCGACCGCGG 903 1937forw CGAACCCCGCCUGGAGGCCGCGG 904 1941forw CCCCGCCUGGAGGCCGCGGUCGG 905 1944rev GGUGCCUCCGGAGAAGCCCCGGG 906 1945rev GGGUGCCUCCGGAGAAGCCCCGG 907 1946forw CCUGGAGGCCGCGGUCGGCCCGG 908 1947forw CUGGAGGCCGCGGUCGGCCCGGG 909 1948forw UGGAGGCCGCGGUCGGCCCGGGG 910 1956rev GCGGUGGCAGUGGGUGCCUCCGG 911 1957forw CGGUCGGCCCGGGGCUUCUCCGG 912 1960forw UCGGCCCGGGGCUUCUCCGGAGG 913 1965rev CAACUCUUCGCGGUGGCAGUGGG 914 1966rev CCAACUCUUCGCGGUGGCAGUGG 915 1986forw CCACUGCCACCGCGAAGAGUUGG 916 1987forw CACUGCCACCGCGAAGAGUUGGG 917 199forw UUUGGAGAAUAAAUUGAAUGAGG 918 1rev CAGAGCCCAACUCUUCGCGGUGG 919 203forw GAGAAUAAAUUGAAUGAGGAAGG 920 203rev CCAUUGCCGGCGAGGGGUGACGG 921 206rev AACUGAUCACCAAAUCUCCAGGG 922 207rev UAACUGAUCACCAAAUCUCCAGG 923 209forw AAAUUGAAUGAGGAAGGCCCUGG 924 209rev AAGCCCCCAUUGCCGGCGAGGGG 925 210rev CAAGCCCCCAUUGCCGGCGAGGG 926 211rev ACAAGCCCCCAUUGCCGGCGAGG 927 216rev GGUUCACAAGCCCCCAUUGCCGG 928 217forw UGAGGAAGGCCCUGGAGAUUUGG 929 217forw GCGCAUCCGUCACCCCUCGCCGG 930 223forw CCGUCACCCCUCGCCGGCAAUGG 931 224forw CGUCACCCCUCGCCGGCAAUGGG 932 225forw GUCACCCCUCGCCGGCAAUGGGG 933 226forw UCACCCCUCGCCGGCAAUGGGGG 934 237rev GCCCAGUCAGUCAGGUUUGGGGG 935 238rev GGCCCAGUCAGUCAGGUUUGGGG 936 239rev UGGCCCAGUCAGUCAGGUUUGGG 937 240rev CUGGCCCAGUCAGUCAGGUUUGG 938 243rev AAGACUUGGCACUUUAUAUGUGG 939 245rev GCACACUGGCCCAGUCAGUCAGG 940 255forw AACCCCCAAACCUGACUGACUGG 941 256forw ACCCCCAAACCUGACUGACUGGG 942 257rev AUAAUCUUGAGUACAAGACUUGG 943 259rev CCUGCCAAUUUGCAGCACACUGG 944 275forw UGGGCCAGUGUGCUGCAAAUUGG 945 279forw CCAGUGUGCUGCAAAUUGGCAGG 946 287forw UACUCAAGAUUAUAAGCAAUAGG 947 28rev CCUCGCCCCCGAGAGACCCGCGG 948 290forw CAAAUUGGCAGGAGACGUGAAGG 949 295rev UUCAUUUUGGCCGACUUUGGAGG 950 298rev CCAUUCAUUUUGGCCGACUUUGG 951 305forw CGUGAAGGCACCUCCAAAGUCGG 952 308rev GGCUCACUGCCCAUUCAUUUUGG 953 318forw CCAAAGUCGGCCAAAAUGAAUGG 954 319forw CAAAGUCGGCCAAAAUGAAUGGG 955 31forw GAGUUGGGCUCUGUCAGCCGCGG 956 329rev GGAACGGCUCCAGGCAACCCCGG 957 32forw AGUUGGGCUCUGUCAGCCGCGGG 958 330forw AAAAUGAAUGGGCAGUGAGCCGG 959 331forw AAAUGAAUGGGCAGUGAGCCGGG 960 331rev UUUCCCCUUCAUAUCUAAGUAGG 961 332forw AAUGAAUGGGCAGUGAGCCGGGG 962 338rev ACCCACGCAGGAACGGCUCCAGG 963 340forw GGCAGUGAGCCGGGGUUGCCUGG 964 345rev CGGGAGAACCCACGCAGGAACGG 965 346forw UAGUGCCUACUUAGAUAUGAAGG 966 347forw AGUGCCUACUUAGAUAUGAAGGG 967 348forw GUGCCUACUUAGAUAUGAAGGGG 968 350rev GAAGACGGGAGAACCCACGCAGG 969 356forw UUAGAUAUGAAGGGGAAAGAAGG 970 356forw UGCCUGGAGCCGUUCCUGCGUGG 971 357forw UAGAUAUGAAGGGGAAAGAAGGG 972 357forw GCCUGGAGCCGUUCCUGCGUGGG 973 364rev GGCAACAAAAAGCGGAAGACGGG 974 365rev AGGCAACAAAAAGCGGAAGACGG 975 372forw AAGAAGGGUUUGAGAUAAUGUGG 976 372rev CCAUAAAAGGCAACAAAAAGCGG 977 373forw AGAAGGGUUUGAGAUAAUGUGGG 978 385rev AGUUGUAAUACAACCAUAAAAGG 979 388forw AAUGUGGGAUGCUAAGAGAAUGG 980 391forw GUGGGAUGCUAAGAGAAUGGUGG 981 392forw CCGCUUUUUGUUGCCUUUUAUGG 982 40forw UCUGUCAGCCGCGGGUCUCUCGG 983 413rev CUCAACAAAAUCUGCAGAGCAGG 984 41forw CUGUCAGCCGCGGGUCUCUCGGG 985 42forw UGUCAGCCGCGGGUCUCUCGGGG 986 434forw CUGCUCUGCAGAUUUUGUUGAGG 987 439forw UUUAGCAUCUACUCUAUGUAAGG 988 43forw GUCAGCCGCGGGUCUCUCGGGGG 989 448rev GACUGGUCGAGAUCUACCUUGGG 990 449rev GGACUGGUCGAGAUCUACCUUGG 991 452forw UGAGGUUUUUGCUUCUCCCAAGG 992 465rev CCACACCCCGUUGAGGGGACUGG 993 470rev UUCUCCCACACCCCGUUGAGGGG 994 471rev GUUCUCCCACACCCCGUUGAGGG 995 472forw AGUGCAAUAGUGCUAAAAACAGG 996 472rev UGUUCUCCCACACCCCGUUGAGG 997 478forw AUCUCGACCAGUCCCCUCAACGG 998 479forw UCUCGACCAGUCCCCUCAACGGG 999 480forw CUCGACCAGUCCCCUCAACGGGG 1000 485forw CCAGUCCCCUCAACGGGGUGUGG 1001 486forw CAGUCCCCUCAACGGGGUGUGGG 1002 488rev CCAGGUUGUAAAGUUUUUUACGG 1003 48forw CCGCGGGUCUCUCGGGGGCGAGG 1004 49forw CGCGGGUCUCUCGGGGGCGAGGG 1005 4rev UGACAGAGCCCAACUCUUCGCGG 1006 506rev UUUCUUUCAUAGCAUCUGCCAGG 1007 508forw CCGUAAAAAACUUUACAACCUGG 1008 530forw GCAGAUGCUAUGAAAGAAAAAGG 1009 531forw CAGAUGCUAUGAAAGAAAAAGGG 1010 532forw AGAUGCUAUGAAAGAAAAAGGGG 1011 536forw GCUAUGAAAGAAAAAGGGGAUGG 1012 537forw CUAUGAAAGAAAAAGGGGAUGGG 1013 549forw AAGGGGAUGGGAGAGAGAGAAGG 1014 54forw GUCUCUCGGGGGCGAGGGCGAGG 1015 552forw GGGAUGGGAGAGAGAGAAGGAGG 1016 553forw GGAUGGGAGAGAGAGAAGGAGGG 1017 561forw UAGAAGAUCUAAAUGAACAUUGG 1018 563forw GAGAGAAGGAGGGAGAGAGAUGG 1019 568forw AAGGAGGGAGAGAGAUGGAGAGG 1020 569forw AGGAGGGAGAGAGAUGGAGAGGG 1021 574rev CAUAAACCGAUGACCAUUAAAGG 1022 57forw AACAAGCGCUAUGACUAGCAAGG 1023 581forw UGGAAAUUGUGUUCCUUUAAUGG 1024 588forw UGUGUUCCUUUAAUGGUCAUCGG 1025 597rev AAAAAGAAACUUCUAACCUCUGG 1026 598forw UUUACUUUUCUUUCAGAUCGAGG 1027 601forw UGGUCAUCGGUUUAUGCCAGAGG 1028 602rev CUCCGUGGAGUUGUCGCUGUCGG 1029 60forw CGGGGGCGAGGGCGAGGUUCAGG 1030 617rev AUUCAGUUAGAUAAACUCCGUGG 1031 620forw GACCGACAGCGACAACUCCACGG 1032 632rev ACUGCUCAAGGUCAUCGCCAAGG 1033 635forw UUUUUUGAAAAAUUAGACCUUGG 1034 63rev UCCUCUUCCUGCGGCCUGAAAGG 1035 644rev GGGUUAUAUCCUACUGCUCAAGG 1036 655forw UGGCGAUGACCUUGAGCAGUAGG 1037 663rev CAGUUUUACAUAUAAAUGACAGG 1038 664rev UUGGAACGCUAAGCUUGUGGGGG 1039 665rev AUUGGAACGCUAAGCUUGUGGGG 1040 666rev UAUUGGAACGCUAAGCUUGUGGG 1041 667rev UUAUUGGAACGCUAAGCUUGUGG 1042 683rev UAUGCCUAGUGUUCCGUUAUUGG 1043 68forw UGACUAGCAAGGUUAAGUGAAGG 1044 690forw CAAGCUUAGCGUUCCAAUAACGG 1045 694forw UAUGUAAAACUGCACUAUACUGG 1046 697rev CCGGCCGCGAAUUUUUAUAAUGG 1047 699forw CGUUCCAAUAACGGAACACUAGG 1048 69forw GGGCGAGGUUCAGGCCUUUCAGG 1049 713forw CUGGCCAUUAUAAAAAUUCGCGG 1050 714forw ACACUAGGCAUAAUGAAAGACGG 1051 716rev CAGGUAUGAGCCACCGCACCCGG 1052 717forw CCAUUAUAAAAAUUCGCGGCCGG 1053 718forw CAUUAUAAAAAUUCGCGGCCGGG 1054 71rev ACUUUAAGCCUUUCAGUCCCUGG 1055 723forw UAAAAAUUCGCGGCCGGGUGCGG 1056 726forw AAAUUCGCGGCCGGGUGCGGUGG 1057 72rev UCGCUCCGUUCCUCUUCCUGCGG 1058 731rev AGGGUUGGGGGUGGGGGGUGUGG 1059 735rev UCCCAAAGUGCUGGGAUUACAGG 1060 736rev CUGGGAGGGUUGGGGGUGGGGGG 1061 737rev GCUGGGAGGGUUGGGGGUGGGGG 1062 738rev GGCUGGGAGGGUUGGGGGUGGGG 1063 739rev CGGCUGGGAGGGUUGGGGGUGGG 1064 73forw AGCAAGGUUAAGUGAAGGCCAGG 1065 740rev CCGGCUGGGAGGGUUGGGGGUGG 1066 743rev CUUCGGCCUCCCAAAGUGCUGGG 1067 743rev CUGCCGGCUGGGAGGGUUGGGGG 1068 744rev GCUUCGGCCUCCCAAAGUGCUGG 1069 744rev ACUGCCGGCUGGGAGGGUUGGGG 1070 745rev GACUGCCGGCUGGGAGGGUUGGG 1071 746rev AGACUGCCGGCUGGGAGGGUUGG 1072 74forw GCAAGGUUAAGUGAAGGCCAGGG 1073 750rev UGGGAGACUGCCGGCUGGGAGGG 1074 751rev GUGGGAGACUGCCGGCUGGGAGG 1075 753forw UACCUGUAAUCCCAGCACUUUGG 1076 754forw ACCUGUAAUCCCAGCACUUUGGG 1077 754rev CUUGUGGGAGACUGCCGGCUGGG 1078 755rev UCUUGUGGGAGACUGCCGGCUGG 1079 757forw UGUAAUCCCAGCACUUUGGGAGG 1080 759rev CAAUUCUUGUGGGAGACUGCCGG 1081 760forw CCACCCCCAACCCUCCCAGCCGG 1082 760rev CUCAAGUGAUCCACCCGCUUCGG 1083 766forw AGCACUUUGGGAGGCCGAAGCGG 1084 767forw GCACUUUGGGAGGCCGAAGCGGG 1085 769rev AAAUCAGAGCCAAUUCUUGUGGG 1086 76forw GUUCAGGCCUUUCAGGCCGCAGG 1087 770forw CUUUGGGAGGCCGAAGCGGGUGG 1088 770rev GAAAUCAGAGCCAAUUCUUGUGG 1089 780forw CGGCAGUCUCCCACAAGAAUUGG 1090 783rev CAGGCUGGUCUCGAACGCCAGGG 1091 784rev CCAGGCUGGUCUCGAACGCCAGG 1092 786forw CGGGUGGAUCACUUGAGCCCUGG 1093 792rev CCAUUAGCUUAUUUUCUUAAAGG 1094 798rev UUUCACCAUGUUGCCCAGGCUGG 1095 802rev GGGGUUUCACCAUGUUGCCCAGG 1096 804forw CCUGGCGUUCGAGACCAGCCUGG 1097 805forw CUGGCGUUCGAGACCAGCCUGGG 1098 812forw CCUUUAAGAAAAUAAGCUAAUGG 1099 813forw CGAGACCAGCCUGGGCAACAUGG 1100 815rev UUUGUUUCUUUCAACCUAGUGGG 1101 816rev GUUUGUUUCUUUCAACCUAGUGG 1102 821forw AAAUAAGCUAAUGGCCCACUAGG 1103 821rev UGUGUUUUUAGUAGAGACGGGGG 1104 822rev UUGUGUUUUUAGUAGAGACGGGG 1105 823rev UUUGUGUUUUUAGUAGAGACGGG 1106 824rev UUUUGUGUUUUUAGUAGAGACGG 1107 82forw GCCUUUCAGGCCGCAGGAAGAGG 1108 839forw CUAGGUUGAAAGAAACAAACAGG 1109 83forw AGUGAAGGCCAGGGACUGAAAGG 1110 842rev GUCGUGAUAAGUGGGCAGAAUGG 1111 850rev AUUACCUUGUCGUGAUAAGUGGG 1112 851rev AAUUACCUUGUCGUGAUAAGUGG 1113 853forw CUAAAAACACAAAAACUAGCUGG 1114 854forw UAAAAACACAAAAACUAGCUGGG 1115 859forw ACACAAAAACUAGCUGGGCGUGG 1116 862forw CAAAAACUAGCUGGGCGUGGUGG 1117 866forw AACUAGCUGGGCGUGGUGGCAGG 1118 866forw UCUGCCCACUUAUCACGACAAGG 1119 871rev UCCUGAGUAGCUGGGAUUACAGG 1120 874rev UUGAAGGUAUGGAUUUGGGACGG 1121 878rev GGAAUUGAAGGUAUGGAUUUGGG 1122 879rev UCUCAGCCUCCUGAGUAGCUGGG 1123 879rev AGGAAUUGAAGGUAUGGAUUUGG 1124 87forw UCAGGCCGCAGGAAGAGGAACGG 1125 880rev GUCUCAGCCUCCUGAGUAGCUGG 1126 885rev AUCCUAAGGAAUUGAAGGUAUGG 1127 890forw GCCUGUAAUCCCAGCUACUCAGG 1128 890rev AGAUGAUCCUAAGGAAUUGAAGG 1129 893forw UGUAAUCCCAGCUACUCAGGAGG 1130 899rev ACUACCCCCAGAUGAUCCUAAGG 1131 903forw AUCCAUACCUUCAAUUCCUUAGG 1132 912forw UUCAAUUCCUUAGGAUCAUCUGG 1133 913forw UCAAUUCCUUAGGAUCAUCUGGG 1134 914forw CAAUUCCUUAGGAUCAUCUGGGG 1135 915forw AAUUCCUUAGGAUCAUCUGGGGG 1136 919rev CACUGCAACCUCUGCCUCCCGGG 1137 920rev UCACUGCAACCUCUGCCUCCCGG 1138 921forw ACACGAGAAUCGCUUGAACCCGG 1139 922forw CACGAGAAUCGCUUGAACCCGGG 1140 924rev CCCCUGGCUGCUCUCUCUCUUGG 1141 925forw GAGAAUCGCUUGAACCCGGGAGG 1142 931forw CGCUUGAACCCGGGAGGCAGAGG 1143 940rev GGCCUUUAUAUACACACCCCUGG 1144 942forw UGCCAAGAGAGAGAGCAGCCAGG 1145 943forw GCCAAGAGAGAGAGCAGCCAGGG 1146 944forw CCAAGAGAGAGAGCAGCCAGGGG 1147 944rev GGAGUCUAGUGGCGUGAUCUCGG 1148 955rev CCAGGCUGGAUGGAGUCUAGUGG 1149 958forw AGCCAGGGGUGUGUAUAUAAAGG 1150 961rev CAGGCUAUCACCCUAAAGGUGGG 1151 962rev UCAGGCUAUCACCCUAAAGGUGG 1152 965rev GCUCUUUCGCCCAGGCUGGAUGG 1153 965rev GAUUCAGGCUAUCACCCUAAAGG 1154 969rev UCUUGCUCUUUCGCCCAGGCUGG 1155 970forw GUAUAUAAAGGCCCACCUUUAGG 1156 971forw UAUAUAAAGGCCCACCUUUAGGG 1157 973rev GGAGUCUUGCUCUUUCGCCCAGG 1158 975forw CCACUAGACUCCAUCCAGCCUGG 1159 976forw CACUAGACUCCAUCCAGCCUGGG 1160 97rev AGGGAAUCGCGCCGCGCGCGGGG 1161 980rev ACUUUCAAUCAUCAGGAUUCAGG 1162 987rev ACUUCUGACUUUCAAUCAUCAGG 1163 98rev CAGGGAAUCGCGCCGCGCGCGGG 1164 994rev AACGAUUUUUUUUUUUGAGACGG 1165 99rev UCAGGGAAUCGCGCCGCGCGCGG 1166

(74) TABLE-US-00004 TABLE4 GuideRNAsequences Seq SeqID Name Sequence NO 1420forw GUGAACCGCGUCUGGUCUGCAGAGGAU 1167 1591rev GCUGACUCUCGCGGCUCUCGUGAGAGU 1168 634forw AACUCCACGGAGUUUAUCUAACUGAAU 1169 640forw ACGGAGUUUAUCUAACUGAAUACGAGU 1170 1688forw CCGGCAGCGCACCGGGUUGCGGAGGGU 1171 716forw GGCCAUUAUAAAAAUUCGCGGCCGGGU 1172 1608forw AGUGACUCUCACGAGAGCCGCGAGAGU 1173 1678forw GCCGACUCGCCCGGCAGCGCACCGGGU 1174 1728rev GGCGCCUACGCCCUUCUCAGUUAGGGU 1175 620forw GGACCGACAGCGACAACUCCACGGAGU 1176 1239rev CAGAAUCUUGUCUCGGCUCAGUGGGAU 1177 1229rev UCUCGGCUCAGUGGGAUGCGUCCGAGU 1178 1584forw CCCCCCAACCAGCCCGCCCGAGAGAGU 1179 481rev GGUUGUAAAGUUUUUUACGGACAGAAU 1180 1625rev AAGGGAGCGGCCGCCGACCGCACGGAU 1181 1896forw CGCCCCUCCCGGGGACCUGCGGCGGGU 1182 1661rev CGCAACCCGGUGCGCUGCCGGGCGAGU 1183 383forw UGAGAUAAUGUGGGAUGCUAAGAGAAU 1184 472forw AAGUGCAAUAGUGCUAAAAACAGGAGU 1185 614rev UAUUCAGUUAGAUAAACUCCGUGGAGU 1186 1376rev UCACCUCGACUACCUUAAAAAUGGAAU 1187 166forw CCAUAAGGAAACUAUGUUAUGAUGAAU 1188 409rev AUAGAGUAGAUGCUAAAUGCUUUGAGU 1189 1170rev UCUCCAGCCUCUCCUUGAGCAGAGGAU 1190 1122rev CUACAUUAUUAAUCUUAAGGACUGAAU 1191 1372forw CUUAGGCCCUAAAAUCUUCCUGUGAAU 1192 268rev AAAUUCCUAUUGCUUAUAAUCUUGAGU 1193 1836rev GCUGACAUUUUUUGUUUGCUCUAGAAU 1194 183forw UAUGAUGAAUGUGAUAGUUUGGAGAAU 1195 1260rev CCCAGGCAGCACUGACUACAGCAGAAU 1196 1034rev GUCUUGAUGAGGUAAAAAGAGGGGAGU 1197 1027forw AAAAAAUCGUUACAAUUUAUGGUGGAU 1198 287forw GUACUCAAGAUUAUAAGCAAUAGGAAU 1199 101rev UUAAUUUCUCUCCUUUGCAUAUUGGAU 1200 1201rev CCUACCCUGCCCCCUUCUCCUUAGAAU 1201 1284forw CUGCUGUAGUCAGUGCUGCCUGGGAAU 1202 429rev CACUUAGCACAGUACCUUACAUAGAGU 1203 355forw ACUUAGAUAUGAAGGGGAAAGAAGGGU 1204 661rev UGCAGUUUUACAUAUAAAUGACAGGAU 1205 1703forw GUUGCGGAGGGUGGGCCUGGGAGGGGU 1206 1332forw AAAAAUGUGAUGAUCAAAACUAGGAAU 1207 1219forw GGCAUUCUAAGGAGAAGGGGGCAGGGU 1208 372forw AAAGAAGGGUUUGAGAUAAUGUGGGAU 1209 192forw UGUGAUAGUUUGGAGAAUAAAUUGAAU 1210 531forw GCAGAUGCUAUGAAAGAAAAAGGGGAU 1211 708rev UAUGAGCCACCGCACCCGGCCGCGAAU 1212 741rev CGCUUCGGCCUCCCAAAGUGCUGGGAU 1213 765forw CCAGCACUUUGGGAGGCCGAAGCGGGU 1214 769forw CACUUUGGGAGGCCGAAGCGGGUGGAU 1215 820rev UUUUGUGUUUUUAGUAGAGACGGGGGU 1216 877rev UGUCUCAGCCUCCUGAGUAGCUGGGAU 1217 886rev CGAUUCUCGUGUCUCAGCCUCCUGAGU 1218 905forw CUACUCAGGAGGCUGAGACACGAGAAU 1219 918rev GCUCACUGCAACCUCUGCCUCCCGGGU 1220 962rev UGCUCUUUCGCCCAGGCUGGAUGGAGU 1221 967rev AGUCUUGCUCUUUCGCCCAGGCUGGAU 1222 991rev UAACGAUUUUUUUUUUUGAGACGGAGU 1223 94rev GCCCAACUCUUCGCGGUGGCAGUGGGU 1224 302rev CCCCAUUGCCGGCGAGGGGUGACGGAU 1225 997rev AACUACCCCCAGAUGAUCCUAAGGAAU 1226 237rev ACUGCAUGUGUGAGCCGAGUCCUGGGU 1227 309rev CACAAGCCCCCAUUGCCGGCGAGGGGU 1228 579forw GAUCUCGACCAGUCCCCUCAACGGGGU 1229 110forw GAGGCACCCACUGCCACCGCGAAGAGU 1230 1082forw GCCCACCUUUAGGGUGAUAGCCUGAAU 1231 456forw GUUGCCUGGAGCCGUUCCUGCGUGGGU 1232 1391rev AAAAAGCGAUCUUAGAUCACCUUGAGU 1233 414forw GCACCUCCAAAGUCGGCCAAAAUGAAU 1234 1182forw CUAUUACCUAAGUAGGUCCCCUGGAAU 1235 192forw GGCCGCAGGAAGAGGAACGGAGCGAGU 1236 875forw UCCCAGCCGGCAGUCUCCCACAAGAAU 1237 1830forw GGUGGUGCAGGGCCGUGAAGCAAGAAU 1238 214rev GGGUGCACGUCCCACAGCUCAGGGAAU 1239 1724rev CAUGCACUUGUCUGUAGUUCAAGGAGU 1240 1070forw GUGUAUAUAAAGGCCCACCUUUAGGGU 1241 337rev CUGGCCCAGUCAGUCAGGUUUGGGGGU 1242 850rev UUGUGGGAGACUGCCGGCUGGGAGGGU 1243 984rev UGAUCCUAAGGAAUUGAAGGUAUGGAU 1244 1086rev UGACUUCUGACUUUCAAUCAUCAGGAU 1245 843rev AGACUGCCGGCUGGGAGGGUUGGGGGU 1246 1014forw UUCAAUUCCUUAGGAUCAUCUGGGGGU 1247 1348forw UGAUUUUGCCAAGAACUUGUCUAGAGU 1248 1703rev AGGAGUCCCCGCCCUUGCAAAAGGGAU 1249 244rev AAAGCGAACUGCAUGUGUGAGCCGAGU 1250 945rev UACCUUGUCGUGAUAAGUGGGCAGAAU 1251 1847forw AAGCAAGAAUGGCCACAGACUGGGAAU 1252 1043forw UUGCCAAGAGAGAGAGCAGCCAGGGGU 1253 50rev GGGCCGACCGCGGCCUCCAGGCGGGGU 1254 131forw AAGAGUUGGGCUCUGUCAGCCGCGGGU 1255 1217forw AGAUACAUUUCUUAGCACUAUUAGAAU 1256 1763rev AGAAGCAAAAGUACCACUAGAUGGAGU 1257 431forw CAAAAUGAAUGGGCAGUGAGCCGGGGU 1258 1322forw GCAUGGUUUUGUGGAAAAGUAAGGAAU 1259 972rev AUUGAAGGUAUGGAUUUGGGACGGAAU 1260 629forw UGAGAGAUCAUUUAACAUUUAAUGAAU 1261 1003forw AAAUCCAUACCUUCAAUUCCUUAGGAU 1262 1244forw UAAGAAAUGUAAAAAAACCUCUAGAGU 1263 836rev CGGCUGGGAGGGUUGGGGGUGGGGGGU 1264 1462forw UCCCACUCUGUCACCCAGAGCUGGAGU 1265 764rev UUAUUGGAACGCUAAGCUUGUGGGGGU 1266 829rev GAGGGUUGGGGGUGGGGGGUGUGGAAU 1267 1445rev CUGCACUCCAGCUCUGGGUGACAGAGU 1268 755forw CUUGGCGAUGACCUUGAGCAGUAGGAU 1269 1436forw UUUCUUCUCUUUCUUUUGAGACGGAGU 1270 1453rev UGGGGACACUGCACUCCAGCUCUGGGU 1271 1544forw UUCUCUCAGCCUCCCAAGUAGCUGGGU 1272 1570rev CUGAAAAUACAAAAAAAUCAGCCGGGU 1273 1621rev GCGGGCGGAUCACCUGAGGUCAGGAGU 1274 1638rev CACUUUGGGAGGCAGAAGCGGGCGGAU 1275 1666forw CGCUUCUGCCUCCCAAAGUGCUGGGAU 1276

(75) In some aspects, a guide RNA molecule can be chemically synthesized using methods standard in the art. In some aspects, a guide RNA molecule can be chemically synthesized such that the guide RNA molecule comprises at least one chemical modification. In some aspects, a guide RNA molecule can be produced by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template, in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods can be performed such that the produced guide RNA molecules comprise at least one chemical modification.

(76) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of the MS2 coat protein (MCP) and at least one transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of the MS2 coat protein (MCP) and at least one transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(77) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(78) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one P65-HSF transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one P65-HSF transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(79) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(80) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one P65-HSF transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one P65-HSF transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(81) In some aspects, any of the compositions of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one VP64 transactivation molecule. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds to the SunTag peptide and at least one VP64 transactivation molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

(82) In some aspects, any composition of the present disclosure can further comprise at least one mRNA and/or polynucleotide encoding at least one rejuvenating factor. In some aspects, the at least one polynucleotide can be a plasmid comprising a nucleic acid encoding at least one rejuvenating factor operably linked to at least one promoter sufficient to drive expression of the at least one rejuvenating factor. A rejuvenating factor can comprise telomerase RNA component (TERC), telomerase associated reverse-transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD/TPP1), TRF-1 interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat binding factor 1 (TRF-1), telomeric repeat binding factor 2 (TRF-2), TERF1 interacting nuclear factor 2 (TIN2), telomeric repeat binding factor 2 (Rap1), Dyskerin Pseudouridine Synthase 1 (DKC1), ribonucleoprotein NHP2 or any combination thereof.

(83) The compositions of the present disclosure can be diluted in at least one cell culture medium. In some aspects, the at least one cell culture medium can comprise adjusted Opti-MEM (Opti-MEM with the pH adjusted to 8.2 or Opti-MEM with the pH adjusted to any value in the range between 7.4 and 8.6), non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS), 1 phosphate-buffered saline (PBS) with the pH in the range between of 7.0 and 8.6 or any combination thereof.

(84) In some aspects, any composition of the present disclosure can be packaged into any cellular delivery system known in the art. Cellular delivery systems can include, but are not limited to, adeno-associated virus (AAV; all serotypes, pseudotypes and hybrids), adenovirus, lentivirus, foamy-virus, herpes simplex virus (HSV) particle, retrovirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measle virus particle, Newcastle disease virus particle, poxvirus particle, picornavirus particle, nanoparticles, exosomes and any combination thereof.

(85) In some aspects, adeno-associate virus can include, but are not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ, AAV-DJ8 or any combination thereof.

(86) The present disclosure provides at least one viral particle, wherein the at least one viral particle comprises any composition of the present disclosure. In some aspects, an at least one viral particle can be an adeno-associated virus (AAV) particle. In some aspects, the at least one viral particle can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV-DJ, or AAV-DJ8 particle. In some aspects, the at least one viral particle can be an adenovirus particle. In some aspects, the at least one viral particle can be a foamy-virus particle. In some aspects, the at least one viral particle can be a lentivirus particle. A retrovirus particle can be MMSV or MSCV particle. A lentivirus particle can be HIV-1 or HIV-2 particle. An alphavirus particle can be SFV, SIN, VEE, or M1 particle. A flavivirus particle can be Kunjin virus, West Nile virus, or Dengue virus particle.

(87) The present disclosure provides at least one exosome, microvesicle or liposome, wherein the at least one exosome, microvesicle or liposome comprises any composition of the present disclosure.

(88) The present disclosure provides at least one nanoparticle, wherein the at least one nanoparticle comprises any composition of the present disclosure. In some aspects, a nanoparticle can comprise a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer based nanoparticle, a metal based nanoparticle, a nanocrystal, a carbon nanotube based nanoparticle or a polymeric micelle. In some aspects, a polymer-based nanoparticle can comprise a multiblock copolymer, a diblock copolymer, a polymeric micelle or a hyperbranched macromolecule. In some aspects, a polymer-based nanoparticle can comprise a multiblock copolymer a diblock copolymer. In some aspects, a polymer-based nanoparticle comprises a poly(lactic-co-glycolic acid) PLGA polymer.

(89) In some aspects, the present disclosure provides a composition comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream of the endogenous hTERC gene.

(90) Kits

(91) In some aspects, the present disclosure provides a kit comprising any composition of the present disclosure. In some aspects, the present disclosure provides a kit comprising any portion of any composition of the present disclosure. In some aspects, any kit of the present disclosure can be used in any method of the present disclosure.

(92) In a non-limiting example, the present disclosure provides a kit comprising a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(93) Rejuvenation Methods of the Present Disclosure

(94) The present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The method can further comprise expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells.

(95) Thus, the present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with a composition comprising a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

(96) The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

(97) The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ; and d) administering the at least one tissue or organ to the subject.

(98) The present disclosure provides a method of producing an in vitro tissue or organ comprising: a) contacting at least one cell with a composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ. The at least one tissue or organ can be used for further in vitro testing, including, but not limited to the testing or drugs and/or therapeutic compounds.

(99) The present disclosure provides a method of producing a plurality of rejuvenated cells comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells.

(100) The present disclosure provides a method of producing a plurality of rejuvenated edited cells comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; and d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cell.

(101) The present disclosure provides a method of treating and/or preventing a disease in a subject comprising: a) contacting a plurality of cells with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

(102) In some aspects, the present disclosure provides a method of treating epidermolysis bullosa (EB) in a subject comprising: a) contacting a plurality of cells comprising keratinocytes, dermal fibroblasts, mesenchymal stem/stromal cells or any combination thereof with a gene editing system such that at least one gene in the genome of at least one cell in the plurality is edited, thereby producing at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the present disclosure; d) expanding the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated edited cells; and e) administering to the subject the plurality of rejuvenated edited cells.

(103) In some aspects, the present disclosure provides a method of rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure. In some aspects, the present disclosure provides a method of rejuvenating at least one cell in a subject comprising administering to the subject at least one therapeutically effective amount of at least one portion of at least one composition of the present disclosure.

(104) In some aspects, the present disclosure provides a method of rejuvenating at least one subject comprising administering at least one therapeutically effective amount of at least one composition of the present disclosure. In some aspects, the present disclosure provides a method of rejuvenating at least one subject comprising administering at least one therapeutically effective amount of at least one portion of at least one composition of the present disclosure.

(105) In some aspects of the methods of the present disclosure, contacting at least one cell with at least one composition of the present disclosure can comprise contacting the at least one cell with a first portion of the at least one composition of the present disclosure and then contacting the at least one cell with a second portion of the least one composition of the present disclosure at least about 1 hour, or at least about 2 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or at least about 7 hours, or at least about 8 hours, or at least about 9 hours, or at least about 10 hours, or at least about 11 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 28 hours, or at least about 32 hours, or at least about 36 hours, or at least about 40 hours, or at least about 44 hours, or at least about 48 hours, or at least about 52 hours, or at least about 56 hours, or at least about 60 hours, or at least about 64 hours, or at least about 68 hours, or at least about 72 hours, or at least about 76 hours, or at least about 80 hours, or at least about 84 hours, or at least about 88 hours, or at least about 92 hours, or at least about 96 hours after contacting the at least one cell with the first portion of the at least one composition of the present disclosure.

(106) Thus, contacting at least one cell with at least one composition of the present disclosure can comprise: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) contacting the at least one cell with at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC), at least about 24 hours after step (a). Optionally, steps (a) and step (b) can be repeated about every 1, or about every 2, or about every 3, or about every 4, or about every 5, or about every 6, or about every 7, or about every 8, or about every 9, or about every 10 days.

(107) In some aspects of the methods of the present disclosure, contacting at least one cell with at least one composition of the present disclosure can further comprise pretreating the at least one cell. In some aspects, pretreating a cell can comprise contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) once about every 4 hours, or about every 8 hours, or about every 12 hours, or about every 16 hours, or about every 20 hours, or about every 24 hours, or about every 28 hours, or about every 32 hours, or about every 36 hours, or about every 40 hours, or about every 44 hours, or about every 48 hours. In some aspects, the at least one cell can be pretreated for at least about 2, or at least about 4, or at least about 6, or at least about 8, or at least about 10 days.

(108) In some aspects, contacting at least one cell with at least one composition of the present disclosure can comprise: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) and at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC); and b) repeating step (a) about every 1, or about every 2, or about every 3, or about every 4, or about every 5, or about every 6, or about every 7, or about every 8, or about every 9, or about every 10 days.

(109) Exemplary transfection regimes are shown in FIGS. 8, 10 and 11.

(110) In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise transfection. In some aspects, transfection can comprise the use of lipofectamine. In some aspects, transfection can comprise any standard transfection method known in the art. In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise electroporation.

(111) In some aspects of the methods of the present disclosure, contacting at least one cell can comprise transfection, transduction, electroporation, nucleofection, at least one cell-penetrating peptide or any combination thereof.

(112) In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise nucleofection. In some aspects, nucleofection can comprise any standard nucleofection method known in the art.

(113) In some aspects of the methods of the present disclosure, contacting at least one cell with a composition of the present disclosure can comprise contacting the cell with at least one cell-penetrating peptides. In some aspects, a cell-penetrating peptide can be an HIV-derived TAT protein. In some aspects, a cell-penetrating peptide can comprise polyarginine. Without wishing to be bound by theory, the at least one cell-penetrating peptide can aid in the delivery of a protein or a RNP complex of the present disclosure to the cytoplasm of a target cell.

(114) In some aspects, at least one composition or at least one portion of at least one composition of the present disclosure can be administered to a subject orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and/or parenterally.

(115) In some aspects of the methods of the present disclosure, expanding at least one cell can comprise culturing the at least one cell using adjusted Opti-MEM, non-adjusted Opti-MEM, human serum, fetal bovine serum (FBS) or any combination thereof.

(116) In some aspect of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERC by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000%, or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell such that the expression level of TERC after contacting the at least one cell with at least one composition of the present disclosure is at least about 0.5 times, or at least about 1.0 times, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times greater as compared to the expression level of TERC prior to contacting the at least one cell with the at least one composition of the present disclosure.

(117) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERC in the at least one cell such that the expression level of TERC is at least about the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times the expression of TERC in a control cell.

(118) In some aspect of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERT by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000% or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%. In some aspects, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell such that the expression level of TERT after contacting the at least one cell with at least one composition of the present disclosure is at least about 0.5 times, or at least about 1.0 times, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times greater as compared to the expression level of TERT prior to contacting the at least one cell with the at least one composition of the present disclosure.

(119) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the expression of TERT in the at least one cell such that the expression level of TERT is at least about the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 1000 times, or at least about 10,000 times, or at least about 20,000 times, or at least about 30,000 times, or at least about 40,000 times, or at least about 50,000 times, or at least about 60,000 times, or at least about 70,000 times, or at least about 80,000 times, or at least about 90,000, or at least about 100,000 times the expression of TERT in a control cell.

(120) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the total number of population doublings exhibited by the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the total number of population doublings exhibited by the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(121) In some aspects, rejuvenating at least one cell can comprise increasing the total number of population doublings such that the number of population doublings exhibited by the at least one cell is at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times or at least about 800 times, or at least about 900 times, or at least about 1,000 times the total number of population doublings exhibited by at least one control cell.

(122) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the length of telomeres in the at least one cell. In some aspects, rejuvenating at least one cell can comprising increasing the length of telomeres in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000%, or at least about 2000%, or at least about 3000%, or at least about 4000%, or at least about 5000%, or at least about 6000%, or at least about 7000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(123) In some aspects, rejuvenating at least one cell can comprise increasing the length of telomeres in the at least one cell such that the length of the telomeres in the at least one cell is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times or at least about 1,000 times the length of telomeres in at least one control cell.

(124) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell by a at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(125) In some aspects, rejuvenating at least one cell can comprise increasing the mitochondrial DNA copy number in the at least one cell such that the mitochondrial DNA copy number is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the mitochondrial DNA copy number in at least one control cell.

(126) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(127) In some aspects, rejuvenating at least one cell can comprise increasing the amount of mitochondrial DNA in the at least one cell such that the amount of mitochondrial DNA in the at least one cell is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the amount of mitochondrial DNA in at least one control cell.

(128) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(129) In some aspects, rejuvenating at least one cell can comprise increasing the number of mitochondria in the at least one cell such that the number of mitochondria is the same as, or at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1,000 times the amount of mitochondria in at least one control cell.

(130) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell. In some aspects, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

(131) In some aspects, rejuvenating at least one cell can comprise increasing the migration activity of the at least one cell such that the migration activity of the at least one cell is the same as, or at least 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, or at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1000 times the migration activity of at least one control cell.

(132) In some aspects, rejuvenating at least one cell can comprise restoring the young-like state of thiol group oxidation on at least one protein in the at least one cell. In a non-limiting example, rejuvenating can comprise increasing the thiol group oxidation of at least one protein in the at least one cell such that the thiol group oxidation of the at least one protein in the at least one cell is comparable to the thiol group oxidation of the same protein in a young cell. In a non-limiting example, rejuvenating can comprise decreasing the thiol group oxidation of at least one protein in the at least one cell such that the thiol group oxidation of the at least one protein in the at least one cell is comparable to the thiol group oxidation of the same protein in a young cell.

(133) In some aspects, rejuvenating at least one cell can comprise decreasing the thiol group oxidation of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%. In some aspects, the at least one protein can be EIF2S1, TM9F3 or USP14.

(134) In some aspects, rejuvenating at least one cell can comprise increasing the thiol group oxidation of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least abouat 100,000%. In some aspects, the at least one protein can be IGFB5.

(135) In some aspects of the methods of the present disclosure, rejuvenating at least one cell can comprise reducing senescence-associated DNA methylation in the at least one cell. In some aspects, reducing senescence-associated DNA methylation in the at least one cell can comprise reducing DNA methylation at least one genomic location which is associated with senescence-related methylation. In some aspects, the at least one genomic location can be cg09780241, cg05099537, cg24541426, cg04316624, cg13180312, cg13316854, cg15726154, cg21507095, cg01697719 or any combination thereof.

(136) In some aspects, rejuvenating at least one cell can comprise reducing DNA methylation at least one genomic location by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%.

(137) In some aspects, an at least one control cell can comprise a cell that has not been contacted with a composition of the present disclosure. In some aspects, an at least one control cell can comprise a cell that has not been contacted with a composition of the present disclosure but has otherwise been grown under the same conditions as at least one cell contacted with a composition of the present disclosure. In some aspects, an at least one control cell can be a dermal fibroblast isolated from a human subject that is 50 years old. In some aspects, an at least one control cell can be a neonatal human epidermal keratinocyte (HEKn). In some aspects, an at least one control cell can be an induced pluripotent stem cell (iPSC).

(138) In some aspects, editing at least one cell can comprise the correction of at least one gene in the at least one cell, the knockout of at least one gene in the at least one cell, the insertion of at least one DNA sequence into the genome of the at least one cell, the deletion of at least one DNA sequence in the genome of the at least one cell or any combination thereof. In some aspects, a gene editing system can comprise any system known in the art for modifying the genome of a target cell, including, but not limited to CRISPR methods, viral methods, etc.

(139) An at least one cell can be obtained and/or isolated from a subject. In some aspects, an at least one cell can be any somatic cell. In some aspects, an at least one cell can be a fibroblast, a keratinocyte, a mesenchymal stem/stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbal stem cell, a retinal pigmented epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a -cell or any combination thereof.

(140) In some aspects, an at least one cell can be an Exocrine secretory epithelial cell, a Brunner's gland cell, an insulated goblet cell of the respiratory and digestive tracts, a stomach foveolar cell, a chief cell, a parietal cell, a pancreatic acinar cell, a paneth cell of small intestine, a Type II pneumocyte of lung, a club cell of lung, a barrier cell, a type I pneumocyte, a gall bladder epithelial cell, a centroacinar cell, an intercalated duct cell, an intestinal brush border cell, a hormone-secreting cell, an enteroendocrine cell, a K cell, an L cell, an I cell, a G cell, an Enterochromaffin cell, an Enterochromaffin-like cell, an N cell, an S cell, a D cell, a Mo cell (or M cell), a Thyroid gland cell, a Thyroid epithelial cell, a Parafollicular cell, a Parathyroid gland cell, a Parathyroid chief cell, an Oxyphil cell, a Pancreatic islets (islets of Langerhans), an Alpha cell, a Beta cell, a Delta cell, an Epsilon cell, a PP cell (gamma cell), an Exocrine secretory epithelial cell, a Salivary gland mucous cell, a Salivary gland serous cell, a Von Ebner's gland cell, a Mammary gland cell, a Lacrimal gland cell, a Ceruminous gland cell, an Eccrine sweat gland dark cell, a Eccrine sweat gland clear cell, an Apocrine sweat gland cell, a Gland of Moll cell, a Sebaceous gland cell, a Bowman's gland cell, an Anterior/Intermediate pituitary cell, a Corticotrope, a Gonadotrope, a Lactotrope, a Melanotrope, a Somatotrope, a thyrotrope, a magnocellular neurosecretory cell, a Parvocellular neurosecretory cell, a Chromaffin cell, an Epithelial cell, a Keratinocyte, an Epidermal basal cell, a Melanocyte, a Trichocyte, a hair shaft cell, a Cortical hair shaft cell, a Cuticular hair shaft cell, a Huxley's layer hair root sheath cell, a Henle's layer hair root sheath cell, an Outer root sheath hair cell, a Surface epithelial cell, a basal cell (stem cell), an Intercalated duct cell, a Striated duct cell, a Lactiferous duct cell, an Ameloblast, an Oral cell, an Odontoblast, a Cementoblast, a neuron, an Auditory inner hair cell, an auditory outer hair cell, a Basal cell of olfactory epithelium cell, a Cold-sensitive primary sensory neuron, a Heat-sensitive primary sensory neuron, a Merkel cell, a Olfactory receptor neuron, a Pain-sensitive primary sensory neuron, a Photoreceptor cell, a Photoreceptor rod cell, a Photoreceptor blue-sensitive cone cell, a Photoreceptor green-sensitive cone cell, a Photoreceptor red-sensitive cone cell, a Proprioceptive primary sensory neuron, a Touch-sensitive primary sensory neuron, a Chemoreceptor glomus cell, an Outer hair cell, an Inner hair cell, a Taste receptor cell, an autonomic neuron, a Cholinergic neuron, a Adrenergic neural cell, a Peptidergic neural cell, an Inner pillar cell, an Outer pillar cell, an Inner phalangeal cell, an Outer phalangeal cell, a Border cell, a Hensen's cell, a Vestibular apparatus supporting cell, a Taste bud supporting cell, a Olfactory epithelium supporting cell, a Schwann cell, a Satellite glial cell, a Enteric glial cell, a glial cell, an interneuron, a Basket cell, a Cartwheel cell, a Stellate cell, a Golgi cell, a Granule cell, a Lugaro cell, a Unipolar brush cell, a Martinotti cell, a Chandelier cell, a Cajal-Retzius cell, a Double-bouquet cell, a Neurogliaform cell, a Retina horizontal cell, an Amacrine cell, a Spinal interneuron, a Renshaw cell, a principal cell, a Spindle neuron, a Fork neuron, a Pyramidal cell, a Place cell, a Grid cell, a Speed cell, a Head direction cell, a Betz cell, a Stellate cell, a Boundary cell, a Bushy cell, a Purkinje cell, a Medium spiny neuron, a Astrocyte (various types), a Oligodendrocyte, a Ependymal cell, a Tanycytes, a Pituicyte, a Lens cell, an Anterior lens epithelial cell, a Crystallin-containing lens fiber cell, a Adipocytes: White fat cell, a Brown fat cell, a Liver lipocyte, a Theca interna cell, a Corpus luteum cell, a Granulosa lutein cell, a Theca lutein cell, a Leydig cell of testes secreting testosterone, a Seminal vesicle cell, a Prostate gland cell, a Bulbourethral gland cell, a Bartholin's gland cell, a Gland of Littre cell, a Uterus endometrium cell, a Juxtaglomerular cell, a Macula densa cell of kidney, a Peripolar cell of kidney, a Mesangial cell of kidney, a barrier cell, a Parietal epithelial cell, a Podocyte, a Proximal tubule brush border cell, a Loop of Henle thin segment cell, a Kidney distal tubule cell, a Kidney collecting duct cell Principal cell, a Intercalated cell, a Transitional epithelium, a Duct cell, a Efferent ducts cell, a Epididymal principal cell, a Epididymal basal cell, a Endothelial cell, a Planum semilunatum epithelial cell, a interdental epithelial cell, a Corneal fibroblasts, a Tendon fibroblasts, a Bone marrow reticular tissue fibroblasts, an Other nonepithelial fibroblasts, a Pericyte Hepatic stellate cell (Ito cell), a Nucleus pulposus cell of intervertebral disc, a Hyaline cartilage chondrocyte, a Fibrocartilage chondrocyte, an Elastic cartilage chondrocyte, a Osteoblast/osteocyte, a Osteoprogenitor cell, a Hyalocyte of vitreous body of eye, a Stellate cell of perilymphatic space of ear, a Pancreatic stellate cell, a Skeletal muscle cell, a Red skeletal muscle cell, a White skeletal muscle cell, an Intermediate skeletal muscle cell, a Nuclear bag cell of muscle spindle, a Nuclear chain cell of muscle spindle, a Myosatellite cell, a Cardiac muscle cell, a Cardiac muscle cell, a SA node cell, a Purkinje fiber cell, a Smooth muscle cell, a Myoepithelial cell, a Erythrocyte, a Megakaryocyte, a Platelets, a Monocyte, a Connective tissue macrophage, a Epidermal Langerhans cell, a Osteoclast, a Dendritic cell, a Microglial cell, a Neutrophil granulocyte, a myeloblast, a promyelocyte, a myelocyte, a metamyelocyte, a Eosinophil granulocyte, a Basophil granulocyte, a Mast cell, a Helper T cell, a Suppressor T cell, a Cytotoxic T cell, a Natural killer T cell, a B cell, a Plasma cell, a Natural killer cell, a Hematopoietic stem cell, a Germ cell, a Oogonium/Oocyte, a Spermatid, a Spermatocyte, a Spermatogonium cell, a Spermatozoon, a Nurse cell, a Granulosa cell, a Sertoli cell, a Epithelial reticular cell, a Interstitial cell, a Interstitial kidney cell or any combination thereof.

(141) In some aspects, a disease can comprise inflammatory disorder, an autoimmune disease, a degenerative disease, cardiovascular disease, ischemic disease, cancer, a genetic disease, a metabolic disorder, idiopathic disorder or any combination thereof. In some aspects, a disease can comprise any medical disorder, including, but not limited to those medical disorders initiated by direct tissue injury (e.g., burns, trauma, decubitus ulcers, etc.), ischemic/vascular events (e.g., myocardial infarct, stroke, shock, hemorrhage, coagulopathy, etc.), infections (e.g., cellulitis, pneumonia, meningitis, SIRS, etc.), neoplasia (e.g., breast cancer, lung cancer, lymphoma, etc.), immunologic/autoimmune conditions (e.g., graft vs. host disease, multiple sclerosis, diabetes, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, etc.), degenerative diseases (e.g., osteoporosis, osteoarthritis, Alzheimer's disease, etc.), congenital/genetic diseases (e.g., epidermolysis bullosa, osteogenesis imperfecta, muscular dystrophies, lysosomal storage diseases, Huntington's disease, etc.), adverse drug effects (e.g., drug-induced hepatitis, drug-induced cardiac injury, etc.), toxic injuries (e.g., radiation exposure(s), chemical exposure(s), alcoholic hepatitis, alcoholic pancreatitis, alcoholic cardiomyopathy, cocaine cardiomyopathy, etc.), metabolic derangements (e.g., uremic pericarditis, metabolic acidosis, etc.), iatrogenic conditions (e.g., radiation-induced tissue injury, surgery-related complications, etc.), and/or idiopathic processes (e.g., amyotrophic lateral sclerosis, Parsonnage-Turner Syndrome, etc.) or any combination thereof. In some aspects, a disease can comprise graft-vs-host diseases (GvHD), Epidermolysis Bullosa (EB), junctional EB (JEB), EB simplex (EBS), congenital ichthyosis, congenital dyskeratosis, Recessive Dystrophic form of EB (RDEB), macular degeneration, Alzheimer's disease, aging, Type II diabetes, heart disease, osteoporosis, chronic skin wounds, diabetes-associated ulcers/wounds, connective tissue diseases such as Ehlers-Danlos Syndrome (EDS) or Marfan syndrome, cancer, or any combination thereof. In some aspects, a disease can also comprise an injury. An injury can comprise a burn, a broken bone, a concussion, a contusion, a fractured bone, a ruptured tendon, a torn ligament, punctured, scarped and/or cut skin, or any other injury known in the art. In some aspects, a disease can be Ehlers-Danlos Syndrome.

(142) As used herein, the term treating or treat describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term treat can also include treatment of a cell in vitro or an animal model.

(143) As used herein, the term preventing, prevent, or protecting against describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

(144) As used herein, the terms ameliorate, ameliorating and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

(145) The terms effective amount and therapeutically effective amount of an agent or compound are used in the broadest sense to refer to a nontoxic but sufficient amount of an active agent or compound to provide the desired effect or benefit.

(146) The term benefit is used in the broadest sense and refers to any desirable effect and specifically includes clinical benefit as defined herein. Clinical benefit can be measured by assessing various endpoints, e.g., inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and/or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; relief, to some extent, of one or more symptoms associated with the disorder; increase in the length of disease-free presentation following treatment, e.g., progression-free survival; increased overall survival; higher response rate; and/or decreased mortality at a given point of time following treatment.

(147) In any method, composition or kit of the present disclosure, TERT can be human TERT (hTERT).

(148) In any method, composition or kit of the present disclosure, TERC can be human TERC (hTERC).

(149) As used herein, a subject includes a mammal. The mammal can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig or any other mammal. In some aspects, a mammal can be a human. The subject can be a male or a female.

EXAMPLES

Example 1Levels of hTERC Transcripts are Higher in Induced Pluripotent Stem Cells as Compared to Fibroblasts

(150) The levels of human telomerase RNA component (hTERC) transcripts in fibroblasts (FBs), induced pluripotent stem cells (iPSCs) were measured using the NanoString nCounter Gene Expression Assay. An approximately 2.4-4.6-fold upregulation of hTERC in iPSCs as compared to the parental F50 (human dermal fibroblast derived from a 50 year old individual) and FN2 (neonatal fibroblasts) lines, as shown in FIG. 4.

Example 2Contacting Somatic Cells with Compositions of the Present Disclosure Increases the Level Of hTERC in the Somatic Cells

(151) In this example, various cell lines were transfected with various compositions of the present disclosure.

(152) Human dermal fibroblasts derived from a 50 year old individual (F50), neonatal human epidermal keratinocytes (HEKn) and GFP-expressing human mesenchymal stem/stromal cells (hMSC-GFP) were subjected to one transfection with 500 ng modified mRNA (mod-mRNA) encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (either independently or as a mix of 4 guides at a 1:1:1:1 ratio) using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). Cells were collected 24 post-transfection. Real time quantitative PCR reactions for human TERC RNA expression were then carried out using the Bio-Rad CFX Connect System. Data was analyzed using the Ct method.

(153) To test the activation of the endogenous hTERC transcript, F50 cells were transfected with either 1 separate individual gRNA (g1, g2, g3, and g4) plus mod-mRNA encoding dCas9-VPR or a mix of all 4 guides at a 1:1:1:1 ratio (gmix) plus mod-mRNA encoding dCas9-VPR. Levels of hTERC transcript were quantified using quantitative reverse transcription PCR. As shown in FIG. 5, the results indicate that even as little as only 1 single guide is sufficient to activate endogenous hTERC (g2, g4) to levels comparable to those achieved by the mix of 4 gRNAs. Transfection with the mix of 4 gRNAs and mod-mRNA encoding dCas9-VPR displayed the greatest increase in expression of hTERC, as shown in FIG. 5.

(154) To monitor endogenous hTERC activation by dCas9-VPR across other cell lines, HEKn and hMSC-GFP cell lines were transfected with a mix of all 4 gRNAs plus mod-mRNA encoding dCas9-VPR. The expression of hTERC was then measured using quantitative reverse transcription PCR. The results are shown in FIG. 6. As shown in FIG. 6, the level of hTERC activation observed in all tested cells lines was comparable to that observed in iPSCs.

Example 2 Methods

(155) Cell lines: 50 year-old human dermal fibroblast (F50, at passage 5), and neonatal human epidermal keratinocytes (HEKn, at passage 7) lines were obtained from ATCC. Human mesenchymal stem/stromal cells with GFP fluorescence (hMSC-GFP, at passage 15) were obtained from Cyagen. The F50 line was cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). HEKn cells were cultured in EpiLife medium supplemented with EDGS and antibiotics (all from ThermoFisher). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen).

(156) Transfections: All transfections of fibroblasts and keratinocytes were performed using Opti-MEMI Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) as a complexation buffer, while transfections of human mesenchymal stem/stromal cells (hMSCs) was performed using Opti-MEM with the pH adjusted to 8.2 (Opti-MEM-pH 8.2) as described in Kogut et al. Nature Communications, 2018. One transfection with 500 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (either individual (g1, g2, g3, g4) or a mix of 4 guides (gmix) at a 1:1:1:1 ratio) or 500 ng modified mRNA encoding dCas9-VPR alone was performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in either Opti-MEM for keratinocyte and fibroblast transfections, or Opti-MEM-pH 8.2 for hMSC transfections. For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA was diluted 10 using either Opti-MEM (for keratinocytes and fibroblasts) or Opti-MEM-pH 8.2 (for hMSCs). After dilution, these components were combined together and incubated for 15 min at room temperature (RT). After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell cultures, in their respective media supplemented with 200 ng/ml B18R (eBioscience).

(157) PCR: F50, HEKn, and hMSC-GFP cells collected 24 hours post-transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript cDNA Synthesis Kit (BioRad). Quantitative PCR (QPCR) reactions for human TERC RNA were performed using SsoAdvanced Universal SYBR Green Supermix. Data was analyzed using the Ct method.

(158) Summary of Example 2: transfecting somatic cells with compositions of the present disclosure, more specifically a mod-mRNA encoding dCas9-VPR in combination with a plurality of gRNAs comprising 1 or more different gRNA species, can increase the expression of hTERC in the transfected cells, including to levels that are comparable to induced pluripotent stem cells.

Example 3Transfecting Mod-RNA Encoding dCas9-VPR Alone does not Induce hTERC Expression in Target Cells

(159) In this example, various cell lines were transfected with various compositions of the present disclosure, specifically a composition comprising only a mod-RNA encoding dCas9-VPR.

(160) 50 year-old human dermal fibroblasts (F50), and GFP-expressing human mesenchymal stem/stromal cells (hMSC-GFP) were subjected to one transfection with 500 ng mod-mRNA encoding dCas9-VPR using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). Cells were collected 24 hours (F50, hMSC-GFP) post-transfection. Quantitative reverse transcription PCR reactions for human TERC RNA expression were carried out using the Bio-Rad CFX Connect System and data was analyzed using the Ct method. The results are shown in FIG. 7. As shown in FIG. 7, the expression levels of hTERC does not increase when only a mod-RNA encoding dCas9-VPR is transfected into a target cell in the absence of any guide RNA.

Example 3 Methods

(161) Cell lines: 50 year-old human dermal fibroblast (F50, at passage 5) were obtained from ATCC. Human mesenchymal stem/stromal cells with GFP fluorescence (hMSC-GFP, at passage p11) were obtained from Cyagen. F50 line was cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen).

(162) Transfections: All transfections of fibroblasts were performed using Opti-MEM as a complexation buffer, while transfections of human mesenchymal stem/stromal cells (hMSCs) was performed Opti-MEM-pH 8.2 One transfection with 500 ng mod-mRNA encoding dCas9-VPR alone was performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in appropriate Opti-MEM. For mod-mRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs was diluted 10 using appropriate Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell cultures, in their respective media supplemented with 200 ng/ml B18R (eBioscience).

(163) PCR: F50 and hMSC-GFP cells were collected 24 hours post-transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript cDNA Synthesis Kit (BioRad). QPCR reactions for human TERC RNA were performed using SsoAdvanced Universal SYBR Green Supermix. Data was analyzed using the 66 Ct method. P values were calculated using a paired, two-tailed Student's t-test. *P0.05, **P0.01, ***P0.001.

(164) Example 3 summary: Increases in hTERC expression using the methods and the compositions of the present disclosure are dependent on specific targeting of a DNA-targeting molecule comprising a transactivation domain, for example through the co-administration of at least one guide RNA.

Example 4The Methods and Compositions of the Present Disclosure Cause an Increase in Population Doubling (PD) of Senescent Fibroblast Cells

(165) In this example, senescent fibroblast cells were contacted with compositions of the present disclosure using methods of the present disclosure.

(166) 50 year-old human dermal fibroblast (F50) line was obtained from ATCC, and subsequently cultured until 90% of cells displayed the senescent phenotype as previously described in Kogut et al, Nature Communications, 2018. Briefly, the senescent phenotype can include an enlargement of cellular morphology and upwards of about 90% positivity for senescence-associated -galactosidase. The F50 line was thawed (F50S, at passage 15, 32.5 PD) and cultured in FEM: DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Initially, 10 k fibroblasts (F50S p15, 32.5 PD) were seeded, per well.

(167) FIG. 8 shows a schematic of the transfection regimen of fibroblasts using rejuvenating compositions of the present disclosure. Initially, 10 k senescent fibroblasts (F50S, p15, 32.5 PD) were seeded, per well. The cells were first pre-treated with three sequential transfections with 500 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(168) The cumulative population doubling of non-treated senescent fibroblasts (F50S, p15, 32.5 starting PD) and the same cells treated with the rejuvenating composition (using the regimen put forth in FIG. 8) was measured. When cells reached 70-80% confluence, they were typsinized, counted using a hemocytometer, and passaged. PD was calculated as the log of the ratio of the final count (N) to the starting (baseline) count (X.sub.0) divided by the log of 2; that is: PD=[log(N X.sub.0)]log 2. P values were calculated using a paired, two-tailed Student's t-test. *P0.05, **P0.01, ***P0.001. The results are shown in FIG. 9. As shown in FIG. 9, the treatment with the rejuvenating composition of the present disclosure results in the increased population doubling.

(169) Example 4 summary: The compositions and methods of the present disclosure can be used to rejuvenate senescent cells, including senescent fibroblasts, leading to an increase in the total number of population doublings exhibited by the treated cells.

Example 5the Methods and Compositions of the Present Disclosure Increase Telomere Length and Mitochondrial DNA Amount in Transfected Target Cells

(170) In this example, various cell lines (low passage and senescent 50 year-old human dermal fibroblasts (F50 and F50S respectively), human mesenchymal stem/stromal cells (hMSCs) and human keratinocytes) were transfected with various compositions of the present disclosure using various methods of the present disclosure. Changes in telomere length in each cell line were then measured.

(171) Low passage and senescent 50 year-old human dermal fibroblasts: 50 year-old human dermal fibroblast (F50) lines were obtained from ATCC, and subsequently cultured until 90% of cells displayed the senescent phenotype as previously described in Kogut et al, Nature Communications, 2018. Breifly, the senescent phenotype can include an enlargement of cellular morphology and upwards of about 90% positivity for senescence-associated -galactosidase. The F50 lined was thawed (F50S, at passage 15, 32.5 PD) and cultured in FEM: DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Initially, 10 k fibroblasts (F50 p3-4 or F50S p15, 32.5 PD) were seeded, per well.

(172) FIG. 8 shows a schematic of a transfection regimen of fibroblasts using a rejuvenating composition of the present disclosure. Initially, 10 k senescent fibroblasts (F50S, p15, 32.5 PD) were seeded, per well. The cells were first pre-treated with three sequential transfections with 500 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(173) FIG. 10 shows a schematic of an alternative transfection regimen of fibroblasts using a rejuvenating composition of the present disclosure. F50 fibroblasts were plated in FEM at 15K cells per well of a 6-well format dish. Four transfection series with 500 ng mod-mRNA encoding hTERT together with 200 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (1 selected guide) were performed every 4 days using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(174) Human mesenchymal stem/stromal cells: Human mesenchymal stem/stromal cells (hMSCs) were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen) under low O.sub.2 (5%). All transfections of hMSCs were performed using Opti-MEM with the pH adjusted to 8.2 (Opti-MEM-pH 8.2) as described in Kogut et al. Nature Communications, 2018.

(175) FIG. 11 shows a schematic of the transfection regimen of hMSCs using rejuvenating compositions of the present disclosure. A pre-treatment of 3 transfections with 500 ng mod-mRNA encoding human TERT was performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). Following pre-treatment with hTERT mod-mRNA transfections, four sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng human TERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM-pH 8.2. For mod-mRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs was diluted 10 using Opti-MEM-pH 8.2. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in respective media supplemented with 200 ng/ml B18R. The medium was changed after overnight incubation after each transfection.

(176) Human keratinocytes: Human neonatal epidermal keratinocytes (HEKn) were cultured in EpiLife medium supplemented with EDGS and antibiotics (all from ThermoFisher). All transfections of HEKs were performed using Opti-MEM with no pH adjustment.

(177) FIG. 11 shows a schematic of the transfection regimen of HEKn using rejuvenating compositions of the present disclosure. A pre-treatment of 3 transfections with 100 ng mod-mRNA encoding human human TERT was performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs was diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in EpiLife medium supplemented with 200 ng/ml B18R (eBioscience). Following pre-treatment of the HEKn cell line, 4 sequential transfection series with 100 ng mod-mRNA encoding hTERT followed by 100 ng mod-mRNA encoding dCas9-VPR+100 ng gRNA mix the next day were performed using LipofectamineRNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM (Thermo Fisher Scientific). For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in EpiLife supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(178) The F5OS cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Quantitative PCR reactions for relative telomere length in treated and untreated cells were performed using SsoAdvanced Universal SYBR Green Supermix. The results are shown in FIG. 12.

(179) The F50 cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the Quick-DNA Miniprep Kit (Zymo Research). Quantitative PCR was used to determine changes in average telomere length in treated and untreated cells based on ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere primer set recognizes and amplifies telomere length by comparing samples to reference genomic DNA containing a 100 base pair (bp) telomere sequence located on human chromosome 17 (provided by kit). Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in FIG. 13.

(180) The hMSCs and HEKn cells were collected 3 days after the last transfection. Genomic DNA (gDNA) was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Quantitative PCR was used to determine changes in average telomere length in treated and untreated cells based on ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere primer set recognizes and amplifies telomere length by comparing samples to reference genomic DNA containing a 100 base pair (bp) telomere sequence located on human chromosome 17 (provided by kit). Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in FIG. 14.

(181) As shown in FIG. 12, FIG. 13 and FIG. 14, F50S, F50, hMSCs and HEKn cells treated with the rejuvenating compositions of the present disclosure displayed increased telomere length as compared to untreated control cells. Moreover, in the case of the treated F50S and HEKn cells, the telomere lengths exceeded the telomere lengths measured in F50-derived induced pluripotent stem cells (F50-iPSCs).

(182) Quantitative PCR was used to determine changes in mitochondrial DNA copy number using ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number Dual Quantification qPCR Assay Kit (#8958). The mtDNA primer set recognizes and amplifies one of the most conserved regions on human mtDNA and will not amplify any off-target sequence on nuclear genomic DNA. The single copy reference (SCR) primer set recognizes and amplifies a 100 bp-long region on human chromosome 17 and serves as reference for data normalization. Primer-probe real-time PCR was performed using BioRad's CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in FIG. 15. As shown in FIG. 15, F50S, hMSCs and HEKn cells treated with the rejuvenating compositions of the present disclosure displayed increased mitochondrial DNA copy number as compared to untreated cells.

(183) Summary of Example 5: the compositions and methods of the present disclosure can be used to rejuvenate various cell types, including low passage and senescent fibroblasts, human mesenchymal stem/stromal cells and human epidermal keratinocytes, leading to an increase in telomere length and mitochondrial DNA amount in treated cells.

Example 6the Methods and Compositions of the Present Disclosure Reactivate Telomerase Activity in Fibroblasts

(184) In this example, 50 year-old human fibroblasts (F50) were transfected with various compositions of the present disclosure. The telomerase activity in the transfected target cells, as well as control cells, was analyzed.

(185) Fifty year-old human dermal fibroblast (F50 passage 6) were cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). As untreated control lines, the F50-iPSC line was cultured in mTeSR1 Media supplemented with 1 mTeSR1 supplement (StemCell Technologies) plus antibiotics (Thermo Fisher Scientific) on plates coated with Matrigel coating matrix (Corning).

(186) Two sequential transfections with either 3 ug mod-mRNA encoding hTERT or 3 ug mod-mRNA encoding hTERT with 3 ug mod-mRNA encoding dCas9-VPR+500 ng gRNA mix were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs was diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(187) Telomerase activity was measured with the TRAPeze Telomerase Detection Kit (Millipore) according to the manufacturer's instructions. CHAPS (1) lysis buffer was used to obtain extracts from, positive control cells (kit provided), an iPSC line derived from F50 (F50-iPSC), fibroblasts (F50), and fibroblasts (F50) treated with two sequential transfections of 3 ug hTERT only or 3 ug hTERT with 3 ug dCas9-VPR+3 ug gRNA mix. About 10,000 cells were assayed for each telomeric repeat amplification protocol assay, and 1,500 cell equivalents were loaded into each well of a 15% non-denaturing TBE (Tris borate, EDTA)-Urea polyacrylamide gel. Each sample was heat inactivated for 10 min at 85 C. to assess the background of the assay.

(188) The results of the telomerase activity assay are shown in FIG. 16. Brighter products are indicative of higher activity. As show in in FIG. 16, the combined treatment with mod-mRNA encoding hTERT, mod-mRNA encoding dCas9-VPR and hTERC-specific gRNAs resulted in a higher level of telomerase activity as compared to untreated iPSCs or F50 cells transfected with only mod-mRNA encoding hTERT treatment alone.

(189) Example 6 summary: The compositions and methods of the present disclosure can reactivate and increase telomerase activity in target cells, thereby rejuvenating the target cells.

Example 7the Compositions of The Present Disclosure Facilitate Single Cell Expansion

(190) In this example, primary human adult fibroblasts were transfected with compositions of the present disclosure to determine if the methods and compositions of the present disclosure could support the expansion from a single cell.

(191) Primary human adult fibroblasts were obtained from a skin biopsy. Adult fibroblasts were cultured in fibroblast expansion medium (FEM) comprised of DMEM/F12 supplemented with 5% human serum, 1 MEM non-essential amino acids solution, 55 M of 2-mercaptoethanol (-ME), 1 GlutaMAX supplement, plus antibiotics (all from Thermo Fisher Scientific), with 50 ug/ml ascorbic acid, 1 ng/ml hydrocortisone (both from Sigma), 12 ng/ml basic FGF (Gibco) and 5 ng/ml human EGF (Invitrogen). Individual patient-derived fibroblasts were plated and single cells selected using a (1010 mm) PYREX cloning cylinder.

(192) FIG. 11 shows a schematic of the transfection regimen of the select, single fibroblasts using rejuvenating compositions of the present disclosure, with adjustments made for the reduced tissue culture surface area. The cells were first pre-treated with three sequential transfections with 50 ng mod-mRNA encoding hTERT. After pretreatment, four sequential transfection series with 50 ng mod-mRNA encoding hTERT followed by 50 ng mod-mRNA encoding dCas9-VPR and 50 ng hTERC guide RNA (gRNA) (4 guides, 1:1:1:1 ratio) the next day were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEMI Reduced Serum Medium (Opti-MEM). For mod-mRNA and/or gRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs and/or gRNA were diluted 10 using Opti-MEM. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and/or gRNA and RNAiMAX were applied to the cell culture, in FEM supplemented with 200 ng/ml B18R (eBioscience). The medium was changed after overnight incubation after each transfection.

(193) Following the transfection regimen, each well was trypsinized and cells were transferred into one well of a 6-well tissue culture plate for further expansion in FEM. As shown in FIG. 17, one week after the last transfection, 2/10 wells of untreated cells expanded successfully and were able to be collected for gDNA extraction while 9/10 wells of treated fibroblasts expanded and collected.

(194) Summary of Example 7: The compositions and methods of the present disclosure can facilitate the expansion of even single cells.

Example 8the Compositions and Methods of the Present Disclosure Increase The Migration Activity of High Passage Human Mesenchymal Stem/Stromal Cells (hMSCs)

(195) In this example, human mesenchymal stem/stromal cells (hMSCs) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can increase the migration activity of high passage hMSCs.

(196) To measure migration activity of treated and untreated hMSCs, a Transendothelial Migration (TEM) assay was used. A schematic of the TEM assay is shown in FIG. 18. Briefly, Corning FluoroBlok cell culture inserts were pre-seeded with human endothelial cells (HUVEC). GFP+hMSCs are plated, and their migration through the HUVEC layer and pores of the FluoroBlok membrane was quantified over time via bottom-reading fluorescence microscopes such as the CellInsight CX7 High-Content Screening (HCS) Platform.

(197) 24-well Corning FluoroBlok Inserts were coated with collagen. After coating, human umbilical vein endothelial cells (HUVECs) were plated at 80K/cm.sup.2 in ECM-2MV BulletKit media (Lonza) and allowed to attach in 5% CO2 incubation overnight. Following overnight incubation and successful attachment, media in the basal chamber was changed to Human Mesenchymal Stem Cell Growth Medium (Cyagen) supplemented with human recombinant EGF [10 ng/mL] (Stemcell Technologies). Human Mesenchymal Stem/Stromal Cells labeled with Green Fluorescent Protein (GFP) purchased from Cyagen were cultured for twelve passages (P12) and treated with the rejuvenating compositions of the present disclosure as described in FIG. 11 and Example 5, while control hMSCs were cultured without mRNA treatment. The rejuvenation procedure lasted for two passages bringing the passage number to P14. A portion of the rejuvenated hMSCs were frozen in a CoolCell LX overnight at 80 while the remaining cells were allowed to remain in culture. After two passages, the frozen cells were thawed and allowed to culture for further two passages.

(198) The four conditions were as follows; Old high passage hMSCs (P20) were never rejuvenated or frozen; Young low passage hMSCs (P5) were a fresh vial of GFP labeled hMSCs (Cyagen) that was thawed allowed to attached overnight then lifted and run on the Transendothelial Migration Assay, frozen rejuvenated hMSCs (P17) that were frozen at P15 then thawed and allowed to culture for two passages and rejuvenated hMSCs that were never frozen but underwent five passages following the rejuvenation protocol. These four conditions were then added to the apical chamber of the FluoroBlok on top of the layer of attached HUVECs. Four fields of view from three replicates were obtained on ThermoScientific's CellInsight CX7 LED High-Content Screening (HCS) Platform. The CX7 HCS is designed to use brightfield, widefield and confocal microscopy for the entire fluorescence spectrum to rapidly capture and quantify high content data such as the kinetic analysis performed on Transendothelial Migration Assays (TEM). As shown in FIG. 19, the rejuvenated high passage hMSCs reached the saturation point significantly faster than untreated young hMSCs (40-50 ordinal time vs 130 ordinal time), while old high passage hMSCs (P20) showed poor migratory ability. A one-way ANOVA analysis exhibited significance of p<0.0001 between young and both groups rejuvenated hMSCs as compared to high passage hMSCs.

(199) Example 8 summary: The compositions and methods of the present disclosure can rejuvenate hMSCs as evidenced by the increase in the migration activity of high passage hMSCs treated using the compositions and methods of the present disclosure.

Example 9the Compositions and Methods of the Present Disclosure Restore the Level of Thiol Group Oxidation of Proteins in High Passage Senescent Human Mesenchymal Stem/Stromal Cells (hMSCs) to that Observed in Young Low Passage hMSCs

(200) In this example, senescent high passage human mesenchymal stem/stromal cells (hMSCs) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can restore the level of thiol group oxidation of proteins in high passage senescent human mesenchymal stem/stromal cells (hMSCs) to that observed in young low passage hMSCs. Among amino acids, the sulphur-containing cysteine (Cys) is particularly prone to oxidation. This is due to the presence of the thiol moiety (SH) in the side chain of Cys, which can easily form disulfide bonds with a different thiol moiety in response to oxidation. Reversible oxidation of Cys thiols regulate the activity of enzymes and ligand binding, as well as participate in redox signaling, which deregulation plays an essential role in the development of many human disease and aging.

(201) Human mesenchymal stem/stromal cells (hMSCs) were cultured in mesenchymal stem cell growth medium (MSCGM) (prepared as a kit from Cyagen) under low O.sub.2 (5%). The three conditions were as follows: Senescent high passage hMSCs (P14) were never rejuvenated; Young low passage hMSCs (P5) were a fresh vial of hMSCs that were thawed allowed to attached overnight then lifted and processed for the peptide analysis; Senescent high passage hMSCs treated at passage 12 with the rejuvenating compositions of the present disclosure. The rejuvenation procedure lasted for two passages bringing the passage number of treated senescent hMSCs to P14 before the peptide analysis was performed, matching untreated senescent hMSCs.

(202) For hMSCS, a pre-treatment of 3 transfections with 500 ng mod-mRNA encoding human TERT was performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). Following pre-treatment with hTERT mod-mRNA transfections, three sequential transfection series with 500 ng mod-mRNA encoding hTERT followed by 500 ng mod-mRNA encoding dCas9-VPR and 500 ng hTERC guide RNA (gRNA) (1 selected guide) the next day were performed using Lipofectamine RNAiMAX (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM-pH 8.2. For mod-mRNA transfections, 100 ng/l RNA was diluted 5, and 5 l of RNAiMAX per microgram of mod-mRNAs was diluted 10 using Opti-MEM-pH 8.2. After dilution, these components were combined together and incubated for 15 min at RT. After incubation at RT, transfection mixtures of mod-RNA mix and RNAiMAX were applied to the cell culture, in respective media supplemented with 200 ng/ml B18R. The medium was changed after overnight incubation after each transfection.

(203) Transfected senescent and un-transfected senescent and low passage young hMSCs were processed using iodoTMTsixplex Isobaric Mass Tag Labeling Kit (ThermoScientific). Resulted iodoTMT labeled peptide mix was analyzed by QExactive HF Orbitrap mass spectrometer with an Easy nLC 1000 UPLC system (Thermo Fischer Scientific). Peptide identifications were performed using MaxQuant program. Each MS/MS spectrum was analyzed against a human specific database (Uniprot). After this analysis, data files were exported and additionally analyzed with Perseus software for data of interest. Each experiment was repeated twice. MaxQuant and Perseus software were downloaded from Max Planck Institute of Biochemistry website.

(204) The level of thiol group oxidation in senescent high passage hMSCs increased in 88 proteins and decreased in 31 proteins as compared to young hMSCs. The transfection of senescent hMSCs with rejuvenating compositions of the present disclosure resulted in the restoration of thiol group oxidation levels in approximately 90% of target proteins to the level observed in young cells. FIG. 20 shows representative results of the thiol group analysis in proteins whose thiol group oxidation levels increased (EIF2S1, TM9F3and USP14) and decreased (IGFB5) in senescent high passage hMSCs and the reversion of these thiol group oxidation levels to the young-like state in response to the treatment with the rejuvenating composition.

(205) Example 9 summary: The compositions and methods of the present disclosure can rejuvenate hMSCs as evidenced by the restoration of the young-like level of protein thiol group oxidation in high passage hMSCs treated using the compositions and methods of the present disclosure.

Example 10the Compositions and Methods of the Present Disclosure Reduce Senescence-Associated DNA Methylation in High Passage Senescent Human Mesenchymal Stem/Stromal Cells (hMSCs) and Human Neonatal Epidermal Keratinocytes (HEKn)

(206) In this example, senescent high passage human mesenchymal stem/stromal cells (hMSCs) and senescent high passage human neonatal epidermal keratinocytes (HEKn) were transfected with compositions of the present disclosure to determine if the compositions and methods of the present disclosure can reduce the level of senescence-associated DNA methylation in these cells. Changes in DNA methylation have been recognized as one of the most common molecular alterations in aging and cellular senescence.

(207) Human fibroblasts of different origin were cultured in FEM; human keratinocytes of different origin were cultured in EpiLife medium supplemented with EDGS and hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM). The following cell types used for DNA methylation analysis were not treated with rejuvenating compositions: young low passage neonatal fibroblasts (P3), young low passage adult F50 fibroblasts (P3), young low passage neonatal keratinocytes HEKn (P3), young low passage fetal keratinocyte (P2), young low passage adult keratinocytes (P3), young umbilical cord-derived hMSCs (P2), senescent high passage F50S fibroblasts (P15), senescent high passage hMSCs (P13) and senescent high passage HEKn (P10). The treated group included senescent high passage HEKn and senescent high passage hMSCs treated with rejuvenating compositions as described in FIG. 11 and Example 5. After completing the treatment with compositions, the treated cells were expanded for additional 6 days. Genomic DNA (gDNA) was extracted from each cell culture using the Quick-DNA Miniprep Kit (Zymo Research) and subjected to DNA methylation analysis using the Illumina Infinium MathylationEPIC BeadChip Kit.

(208) The DNA methylation data generated for cells of different types were analyzed using the R package IlluminaHumanMethylationEPICanno.ilm10b2. hg19 and combined into three groups as follows: young cells, senescent (high passage) cells and senescent cells treated with rejuvenating compositions of the present disclosure. The young group included young low passage neonatal fibroblasts (P3), young low passage adult fibroblasts (P3), young low passage neonatal keratinocytes (P3), young low passage fetal keratinocyte (P2), young low passage adult keratinocytes (P3) and young umbilical cord-derived hMSCs (P2). The senescent group included senescent high passage F50S fibroblasts (P15), senescent high passage hMSCs (P13) and senescent high passage HEKn (P10). The treated group included senescent high passage HEKn and senescent high passage hMSCs treated with rejuvenating compositions. Cells of different type and origin were combined based on their senescence state to eliminate cell type-specific methylation differences among groups. The groups were compared using the 2-tailed t-test for two groups with unequal variance, and the degree of methylation was calculated as a fraction of methylated nucleotides at a site of interest, ranging from 0 to 1. Differential methylation sites were selected based on the largest difference in the degree of methylation for each group, and 9 DNA methylation sites were identified as the most methylated in senescent high passage cells irrespectively of the cell type of origin.

(209) FIG. 21 depicts the location of 9 identified senescence-associated DNA marks and their associated genomic loci. All 9 sites showed an increase in DNA methylation levels in the senescence high passage group. The treatment of the cells from the senescent group with rejuvenating compositions of the present disclosure reduced the level of DNA methylation at all 9 sites to the level similar to that of the young cell group.

(210) Example 10 summary: the compositions and methods of the present disclosure can be used to rejuvenate various cell types, including senescent human mesenchymal stem/stromal cells and human epidermal keratinocytes, leading to a reduction in senescence-association DNA methylation in treated cells.