Anc80 encoding sphingolipid-metabolizing proteins

Abstract

The present disclosure pertains to the use of an Anc80 viral vector that encodes a sphingolipid-metabolizing protein such as acid ceramidase to achieve expression of the sphingolipid-metabolizing protein in a mammalian cell or group of cells. Expression of the protein from the Anc80 vector reduces high levels of ceramide in the cell that lead to cell death or senescence.

Claims

1. A method to restore heart function in a subject following ischemia, reperfusion injury or myocardial infarction (MI), the method comprising: directly administering a therapeutically effective amount of an Anc80 viral vector comprising a nucleotide sequence encoding an acid ceramidase protein to cardiac cells of the subject in vivo, wherein the nucleotide sequence encoding the acid ceramidase is operatively linked to an expression control sequence.

2. The method of claim 1, wherein the nucleotide sequence encoding the acid ceramidase is as set out in one of SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 7.

3. The method of claim 1, wherein the nucleotide sequence encoding the acid ceramidase is as set out in SEQ ID NO: 1.

4. The method of claim 1, wherein the nucleotide sequence encoding the acid ceramidase is as set out in SEQ ID NO: 6.

5. The method of claim 1, wherein the nucleotide sequence encoding the acid ceramidase is as set out in SEQ ID NO: 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1E show the characterization of cell death dynamics and sphingolipid-metabolizing enzymes expression in mouse heart after MI. Hearts were harvested from sham operated mice or 4 hours 1, 2, 4 and 28 days post MI. A) TUNEL stain was used to assess DNA fragmentation in cardiac cells in non-treated, 1, 2, 4 and 28 days post MI. Troponin-I immunostaining was used to distinguish between cardiomyocytes and non-cardiomyocytes. B) Dendogram of Sphingolipids signaling pathway transcriptome in sham hearts, 4 h and 24 h post ligation. C) Acid Ceramidase (AC), Sphk1 and S1PR2 mRNA levels relative to 18s rRNA was assessed in the left ventricle (LV) in early stages of MI development by quantitative PCR D) Protein levels of AC and Sphk1 were assessed in the LV at early stages of MI development by western-blot. E) AC activity in the LV after MI at early stages of MI development.

(2) FIGS. 2A-2E show the characterization of cell death dynamics and sphingolipid-metabolizing enzymes expression in mouse heart after MI. A) Dendogram of Sphingolipids metabolism genes transcriptome in sham hearts, at 4 and 24 hours post ligation. B) Volcano plots of Sphingolipids metabolism genes transcriptome and sphingolipids signaling pathway transcriptome, 4 and 24 hours. C) Protein levels of pro-caspase and cleaved caspase in the LV of sham hearts and hearts 24 hours post MI. D) Protein levels of Sphk1 and -actin in sham hearts and hearts 4 and 24 hours post MI. E) Protein levels of S1PR2 and -actin in sham hearts and hearts 4 and 24 hours post MI.

(3) FIG. 3 shows EGFP expression dynamics after direct injection into the heart of 2.510.sup.11 genome copies (GC) of Anc80 encoding EGFP.

(4) FIG. 4 Luciferase (Luc) expression dynamics after direct injection to the heart of Anc80 or AAV9 encoding firefly luciferase.

(5) FIG. 5 shows EGFP expression dynamics post infection with 110.sup.10 GC/110.sup.6 cells of Anc80 or AAV9 encoding EGFP. Neonatal rat cardiomyocytes were infected with 1.2510.sup.10 GC of Anc80 (A, B, C) or AAV9 (D, E, F) encoding EGFP. The cells were imaged using a fluorescence microscope 18 hours (A, D) 48 hours (B, E) and 96 hours (C, F) post infection.

(6) FIG. 6 show EGFP expression dynamics after IM injection of 2.510.sup.11 GC of Anc80 or AAV9 encoding EGFP. Adult rat hearts were injected intramyocardially (IM) with 2.510.sup.11 GC of Anc80 (A, B) or AAV9 (C, D) encoding EGFP. The hearts were collected 1 day (A, C) and 45 days (B, D) post injection. Heart sections were imaged using a fluorescence microscope.

(7) FIG. 7 shows the biodistribution of Anc80 and AAV9 in a rat model (IVIS). Adult rat hearts were injected intramyocardially (IM) with 7.510.sup.10 GC of Anc80 (A, B, C, D) or AAV9 (E, F, G, H) encoding to firefly luciferase. Bioluminescence was measured using a IVIS machine 24 hours (A, E), 72 hours (B, F) 1 week (C, G) and 4 weeks (D, H) post injection. I) Luciferase activity kinetics as measured using an IVIS machine.

(8) FIG. 8 shows EGFP expression 6 weeks after IM injection of 2.510.sup.11 GC of Anc80 or AAV9 encoding EGFP. Adult rat hearts were injected intramyocardially (IM) with 2.510.sup.11 GC of Anc80 (A) or AAV9 (B) encoding EGFP. The hearts were collected 6 weeks post injection and sectioned. Heart sections were imaged using a fluorescence scanner.

(9) FIG. 9 shows EGFP expression 6 weeks after IC injection of 2.510.sup.11 GC of Anc80 encoding EGFP. Adult rat heart was injected intracoronary (IC) with 2.510.sup.11 GC of Anc80 encoding EGFP. The heart was collected 6 weeks post injection. Heart sections were imaged using a fluorescence scanner.

(10) FIG. 10 shows EGFP biodistribution. Adult rat hearts were injected intramyocardially (IM) with 2.510.sup.11 GC of AAV9 (A, B) or Anc80 (C, D) encoding EGFP. The hearts were collected 6 weeks post injection. Heart sections were immunostained with GFP antibody and a cardiomyocyte-specific marker, -actinin antibody. (E) GFP expression in heart liver and lung was assessed using western blot analysis.

(11) FIG. 11 shows Anc80 viral genome bio-distribution 8 weeks post IM injection. Adult rats hearts were injected intramyocardially (IM) with 7.510.sup.10 GC of Anc80 encoding firefly luciferase. Viral genome copies in the heart, lung, liver, brain and spinal cord were assessed using qPCR using firefly luciferase specific primers.

(12) FIGS. 12A and 12B show results of a sheep model of myocardial infarction and gene delivery of AC by anc80 vector. T2 maps work by tracking signal of water molecules and granulocytes linked to inflammation. Higher hotter T2 times indicate inflammation. The AC animal in area of injection has purple normal T2 times in fully rescued myocardium which should be infarcted. Damage that accrued in the lower non injected slice can be seen. AC has effects on both viability and reducing inflammation from the infarction. (A) T2 MRI mapped image featuring inflammation response in the lower apex portion of the heart from a gene therapy treated animal. Higher T2 values indicate active damage and myocardium at risk, in this case 90-100 consistent with detectable infarction. (B) T2 MRI mapped image from an ovine subject overexpressing AC gene therapy at 4 weeks. Normal T2 times of 40-50 (purple) indicating robust healthy myocardium in the area of injections and surrounding myocardium.

(13) FIGS. 13A and 13B are a series of stills from a movie (A) showing a 3 month post infarction animal treated with AAV control or non-expression null vector. This figure shows that there is profound diskinesis and bulging in the middle to upper portions of the wall. Infarcted dead heart tissue bulges and does not contribute to the contraction. (B) The first AC animal demonstrated robust rescue of function from the effects of the MI. There is nice strong contraction profile all around the heart slices that were injected. In the lower non injected area bulging can be seen. As shown, this area is much less in terms of infarction size and impact on overall function. This animal had a score of 60% ejection fraction which is basically normal. At 4 weeks the animals usually drop to 45-53% range and by 3 months drop in the 35-40% range as in the control video. Ejection fraction is the main endpoint to assess function. Additionally, scar size from the MI was only 5% compared to controls in which scar size is usually in the range of 13-25%.

(14) FIG. 14 is a map of an Anc80.AC vector used in the method of the disclosure.

(15) FIG. 15 shows western blot analysis of AC expression post AC CMV vector injection into mouse cochlear using AC H-41 antibody.

(16) FIG. 16 shows the vector map of the AC CMV vector that was injected into mouse cochlear tissue.

DETAILED DESCRIPTION OF THE DISCLOSURE

(17) All patents, published applications and other references cited herein are hereby incorporated by reference into the present application.

(18) In the description that follows, certain conventions will be followed as regards the usage of terminology. In general, terms used herein are intended to be interpreted consistently with the meaning of those terms, as they are known to those of skill in the art. Some definitions are provided purely for the convenience of the reader.

(19) The term cell or group of cells is intended to encompass single cells as well as multiple cells either in suspension or in monolayers. Whole tissues also constitute a group of cells.

(20) The term cell quality or quality of a cell refers to the level of cell viability, and cellular function of a cell as measured against a normal healthy cell of the same type with normal cell function and expected life span, the quality of cells that are programmed for survival but not for cell death. Embryo quality is the ability of an embryo to perform successfully in terms of conferring a high pregnancy rate and/or resulting in a healthy offspring and is assessed mainly by microscopic evaluation at certain time points following in vitro fertilization. Embryo profiling is the estimation of embryo quality by qualification and/or quantification of various parameters known to those of skill in the art including but not limited to number of pronuclei, cell number, cell regularity, degree of fragmentation. Estimations of embryo quality guides the choice in embryo selection in in vitro fertilization.

(21) The term inhibit or inhibition when used in conjunction with senescence includes the ability of the sphingolipid-metabolizing proteins of the disclosure to reverse senescence, thereby returning to normal or near normal function.

(22) The terms stress, stress-related events or cellular-stress refers to a wide range of molecular changes that cells undergo in response to environmental stressors, such as extreme temperatures, exposure to toxins, mechanical damage, anoxia, and noise.

(23) The term robustness as it is used herein, refers to the quality or condition of being strong and in good condition with normal function.

(24) The present technology is based on the use of sphingolipid metabolizing protein in order to manipulate the fate of cells post stress-related events and during aging. Different types of stress can initiate the signal transduction that leads to two major pathways: one can lead to cell death and the other leads to senescence, which is characterized by low cell function and arrested regeneration and amplification. In addition, senescent cells secrete different factors that can trigger the immune response and lead to inflammation and additional cell death. Cell senescence can be initiated not only by stress but also during aging. Both the cell death and cell senescence pathways involve sphingolipid metabolism mainly an increase in ceramide that can lead to both.

(25) Ceramide has been shown to induce apoptotic cell death in different cells type including murine and human cardiomyocytes. On the other hand, sphingosine, one of the products of ceramide degradation can be phosphorylated to give rise to a major agent of cell survival and cardioprotection, sphingosine 1 phosphate.

(26) There are several studies that support association of the signaling lipid, ceramide, and its metabolizing enzymes with cellular and organismal aging. It has been reported that the intracellular level of ceramide increased during stress related signaling such as cell culture and aging. Ceramidase, for example, acid ceramidase (AC) is required to hydrolyze ceramide into sphingosine and free fatty acids. Sphingosine is rapidly converted to sphingosine-1-phosphate (S1P), another important signaling lipid that counteracts the effects of ceramide and promotes cell survival. Thus, AC is a rheostat that regulates the levels of ceramide and S1P in cells, and as such participates in the complex and delicate balance between death and survival.

(27) For example, we have previously shown that AC expression is carefully regulated during oocyte maturation and early embryo development (Eliyahu, et al, 2010). We have also found that the complete knock-out of AC function in mice leads to embryo death between the 2 and 8-cell stage (Eliyahu, FASEB J, 2007). In addition, our previous publication (Eliyahu, FASEB J, 2010) showed that the ceramide-metabolizing enzyme, AC is expressed and active in human cumulus cells and follicular fluid, essential components of this environment, and that the levels of this enzyme are positively correlated with the quality of human embryos formed in vitro. These observations led to a new approach for oocyte and embryo culture that markedly improves the outcome of in vitro fertilization (IVF).

(28) In this disclosure, we describe a strategy different from previously described approaches to reduce ceramide levels in the ischemic heart. Instead of targeting ceramide synthesis, we study the effect of increasing ceramide hydrolysis by overexpression of acid ceramidase. With this strategy, not only can we reduce ceramide levels but we also increase the reservoir of sphingosine which is the main building block for the pro-survival molecule sphingosine-1-phosphate (S1P).

(29) Choice of Vehicle and Duration of Expression Needed

(30) Methods and compositions for in vivo delivery of acid ceramidase that express a sphingolipid-metabolizing protein such as ceramidase were explored. Each provides a different duration of ceramidase expression depending on the time that expression is needed given the particular situation. For example, use of the protein form is suitable if short term activity is required for up to 72 hours, mainly for in vitro applications, cell culture, cell therapy, or during primary or stem cell derivation. Use of the protein form is generally applicable to any cell type in vivo, including gametes. Moreover, dermatological applications, such as anti-aging treatments for the skin, lend themselves to use of the protein.

(31) For applications where more sustained expression of a sphingolipid metabolizing enzyme is required, for example in a method for restoring cardiac function following a myocardial infarction, hearing and vision loss, orthopedic and neuronal injuries and the like, expression from an Anc80 vector may be desirable.

(32) Adeno-associated viruses have emerged as one of the most promising vectors in the field of gene therapy. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAVs) as a safe and efficient delivery vehicle for gene transfer. AAV vectors are known to be expressed for several months or longer post administration; thus, they provide a more extensive time frame than modRNA.

(33) More recently, Zinn et al. identified Anc80 as a highly potent in vivo gene therapy vector for targeting liver, muscle and retina. Anc80 virus, an in silico designed gene therapy vector, has demonstrated high gene expression levels in the liver, eye and ear compared to naturally occurring adeno-associated viral vectors (AAVs) that are currently in clinical development. Due to its synthetic nature, Anc80 does not circulate in humans, making it less likely to be recognized immunologically by antibodies against naturally-occurring AAVs. Anc80 also provides longer lasting expression. In addition, Anc80 expresses protein in much higher amounts than AAVs, so the amount of necessary virus is much less that leads to lower immune response.

(34) The present disclosure, therefore, also provides a method for inhibiting or reducing damage to cardiac cells following MI by administration of a cocktail of Anc80 virus encoding sphingolipid metabolizing proteins. The treatment includes different combinations of Acid Ceramidase (AC) and/or Sphingosine Kinase (SPHK) and/or Sphingosine-1-phosphate receptor (S1PR) gene (cDNA). Anc80 virus, an in silico designed gene therapy vector, Anc80 has demonstrated high gene expression levels in the liver, eye and ear compared to naturally-occurring adeno-associated viral vectors (AAVs) that are currently in clinical development. Anc80, an engineered gene therapy vector, is synthetic in nature and has been shown to reduce cross-reactivity with commonly used AAV vectors. Anc80 is a potent gene therapy vector that is not known to circulate in humans, making it less likely to cross-react immunologically with naturally occurring AAVs.

(35) Sphingolipid-Metabolizing Proteins

(36) In one embodiment, a composition useful for practicing the method of the present disclosure may include either individually or in different combinations Anc80 vectors encoding the following sphingolipid-metabolizing proteins: ceramidase (acid, neutral or alkaline), sphingosine kinase (SPHK), sphingosine-1-phosphate receptor (S1PR), and a ceramide kinase (CERK). In one embodiment, the sphingolipid-metabolizing protein is a ceramidase.

(37) Ceramidase is an enzyme that cleaves fatty acids from ceramide, producing sphingosine (SPH), which in turn is phosphorylated by a sphingosine kinase to form sphingosine-1-phosphate (S1P). Ceramidase is the only enzyme that can regulate ceramide hydrolysis to prevent cell death and SHPK is the only enzyme that can synthesize sphingosine 1 phosphate (S1P) from sphingosine (the ceramide hydrolysis product) to initiate cell survival. S1PR, a G protein-coupled receptor binds the lipid-signaling molecule S1P to induce cell proliferation, survival, and transcriptional activation. CERK is an phosphatase that phosphorylates ceramide into ceramide 1 phosphate to induce cell survival.

(38) Presently, 7 human ceramidases encoded by 7 distinct genes have been cloned: acid ceramidase (ASAH1)associated with cell survival; neutral ceramidase (ASAH2, ASAH2B, ASAH2C)protective against inflammatory cytokines; alkaline ceramidase 1 (ACER1)mediating cell differentiation by controlling the generation of SPH and S1P; alkaline ceramidase 2 (ACER2)important for cell proliferation and survival; and alkaline ceramidase 3 (ACER3).

(39) The nucleotide sequences for nucleic acids encoding these ceramidases are shown in Table 1.

(40) In one embodiment, Anc80, a relatively nascent technology, has shown considerable potential as a delivery vehicle for gene therapy in disease, for example, cardiac disease, hearing loss, vision loss and neurodegenerative diseases. Anc80 as an engineered gene therapy vector is synthetic in nature and is not known to circulate in humans. It has been shown to have reduced cross-reactivity with commonly used AAV vectors. Anc80 therefore is a potent gene therapy vector, which is less likely to be recognized immunologically by antibodies against naturally occurring AAVs.

(41) An Anc80 vector encoding acid ceramidase (Anc80.AC) has multiple advantages over other potential anti-apoptotic factors.

(42) Low Toxicity

(43) Physiological enzymes are expected to have no toxic effects. The AC protein is present as two forms (active and inactive) in the cell. The inactive AC protein undergoes an auto-self cleavage to the active form, which is responsible for hydrolyzing ceramide to sphingosine after exposure to stress. Transfecting cells with Anc80.AC increases mostly the inactive precursor of the enzyme; this allows physiological control to regulate the amount of active AC protein required for survival. AC should not influence other cellular signaling because the only known biological function of AC is the control of ceramide metabolism. The creation of a mouse model in which the AC enzyme (COEAC) is constantly overexpressed in all tissues demonstrates a lack of toxicity as the result of AC overexpression.

(44) Unique Physiological Function of Acid Ceramidase

(45) Increase in ceramide level can have different outcomes leading to cell death and/or senescence. Ceramidase is the only enzyme that can hydrolyze ceramide and hydrolysis of ceramide is its putative function.

(46) Table 1 contains the nucleotide sequences to be encoded by the vectors disclosed for use in practicing the method.

(47) TABLE-US-00001 TABLE1 Gene OpenReadingFrame ASAH1 ATGCCGGGCCGGAGTTGCGTCGCCTTAGTCCTCCTGGCTGCCGCCGTCAGCTGTGCCGTCGCGCA transcript GCACGCGCCGCCGTGGACAGAGGACTGCAGAAAATCAACCTATCCTCCTTCAGGACCAACGTAC variant1 AGAGGTGCAGTTCCATGGTACACCATAAATCTTGACTTACCACCCTACAAAAGATGGCATGAATT (ACv1) GATGCTTGACAAGGCACCAGTGCTAAAGGTTATAGTGAATTCTCTGAAGAATATGATAAATACAT TCGTGCCAAGTGGAAAAATTATGCAGGTGGTGGATGAAAAATTGCCTGGCCTACTTGGCAACTTT CCTGGCCCTTTTGAAGAGGAAATGAAGGGTATTGCCGCTGTTACTGATATACCTTTAGGAGAGAT TATTTCATTCAATATTTTTTATGAATTATTTACCATTTGTACTTCAATAGTAGCAGAAGACAAAAAA GGTCATCTAATACATGGGAGAAACATGGATTTTGGAGTATTTCTTGGGTGGAACATAAATAATGA TACCTGGGTCATAACTGAGCAACTAAAACCTTTAACAGTGAATTTGGATTTCCAAAGAAACAACA AAACTGTCTTCAAGGCTTCAAGCTTTGCTGGCTATGTGGGCATGTTAACAGGATTCAAACCAGGA CTGTTCAGTCTTACACTGAATGAACGTTTCAGTATAAATGGTGGTTATCTGGGTATTCTAGAATGG ATTCTGGGAAAGAAAGATGTCATGTGGATAGGGTTCCTCACTAGAACAGTTCTGGAAAATAGCA CAAGTTATGAAGAAGCCAAGAATTTATTGACCAAGACCAAGATATTGGCCCCAGCCTACTTTATC CTGGGAGGCAACCAGTCTGGGGAAGGTTGTGTGATTACACGAGACAGAAAGGAATCATTGGAT GTATATGAACTCGATGCTAAGCAGGGTAGATGGTATGTGGTACAAACAAATTATGACCGTTGGA AACATCCCTTCTTCCTTGATGATCGCAGAACGCCTGCAAAGATGTGTCTGAACCGCACCAGCCAA GAGAATATCTCATTTGAAACCATGTATGATGTCCTGTCAACAAAACCTGTCCTCAACAAGCTGACC GTATACACAACCTTGATAGATGTTACCAAAGGTCAATTCGAAACTTACCTGCGGGACTGCCCTGA CCCTTGTATAGGTTGGTGA(SEQIDNO:1) Sphk1 ATGGATCCAGTGGTCGGTTGCGGACGTGGCCTCTTTGGTTTTGTTTTCTCAGCGGGCGGCCCCCG GGGCGTGCTCCCGCGGCCCTGCCGCGTGCTGGTGCTGCTGAACCCGCGCGGCGGCAAGGGCAA GGCCTTGCAGCTCTTCCGGAGTCACGTGCAGCCCCTTTTGGCTGAGGCTGAAATCTCCTTCACGCT GATGCTCACTGAGCGGCGGAACCACGCGCGGGAGCTGGTGCGGTCGGAGGAGCTGGGCCGCTG GGACGCTCTGGTGGTCATGTCTGGAGACGGGCTGATGCACGAGGTGGTGAACGGGCTCATGGA GCGGCCTGACTGGGAGACCGCCATCCAGAAGCCCCTGTGTAGCCTCCCAGCAGGCTCTGGCAAC GCGCTGGCAGCTTCCTTGAACCATTATGCTGGCTATGAGCAGGTCACCAATGAAGACCTCCTGAC CAACTGCACGCTATTGCTGTGCCGCCGGCTGCTGTCACCCATGAACCTGCTGTCTCTGCACACGGC TTCGGGGCTGCGCCTCTTCTCTGTGCTCAGCCTGGCCTGGGGCTTCATTGCTGATGTGGACCTAG AGAGTGAGAAGTATCGGCGTCTGGGGGAGATGCGCTTCACTCTGGGCACCTTCCTGCGTCTGGC AGCCCTGCGCACCTACCGCGGCCGACTGGCCTACCTCCCTGTAGGAAGAGTGGGTTCCAAGACAC CTGCCTCCCCCGTTGTGGTCCAGCAGGGCCCGGTAGATGCACACCTTGTGCCACTGGAGGAGCCA GTGCCCTCTCACTGGACAGTGGTGCCCGACGAGGACTTTGTGCTAGTCCTGGCACTGCTGCACTC GCACCTGGGCAGTGAGATGTTTGCTGCACCCATGGGCCGCTGTGCAGCTGGCGTCATGCATCTGT TCTACGTGCGGGCGGGAGTGTCTCGTGCCATGCTGCTGCGCCTCTTCCTGGCCATGGAGAAGGG CAGGCATATGGAGTATGAATGCCCCTACTTGGTATATGTGCCCGTGGTCGCCTTCCGCTTGGAGC CCAAGGATGGGAAAGGTGTGTTTGCAGTGGATGGGGAATTGATGGTTAGCGAGGCCGTGCAGG GCCAGGTGCACCCAAACTACTTCTGGATGGTCAGCGGTTGCGTGGAGCCCCCGCCCAGCTGGAA GCCCCAGCAGATGCCACCGCCAGAAGAGCCCTTATGA(SEQIDNO:2) S1PR2 ATGGGCAGCTTGTACTCGGAGTACCTGAACCCCAACAAGGTCCAGGAACACTATAATTATACCAA GGAGACGCTGGAAACGCAGGAGACGACCTCCCGCCAGGTGGCCTCGGCCTTCATCGTCATCCTCT GTTGCGCCATTGTGGTGGAAAACCTTCTGGTGCTCATTGCGGTGGCCCGAAACAGCAAGTTCCAC TCGGCAATGTACCTGTTTCTGGGCAACCTGGCCGCCTCCGATCTACTGGCAGGCGTGGCCTTCGT AGCCAATACCTTGCTCTCTGGCTCTGTCACGCTGAGGCTGACGCCTGTGCAGTGGTTTGCCCGGG AGGGCTCTGCCTTCATCACGCTCTCGGCCTCTGTCTTCAGCCTCCTGGCCATCGCCATTGAGCGCC ACGTGGCCATTGCCAAGGTCAAGCTGTATGGCAGCGACAAGAGCTGCCGCATGCTTCTGCTCATC GGGGCCTCGTGGCTCATCTCGCTGGTCCTCGGTGGCCTGCCCATCCTTGGCTGGAACTGCCTGGG CCACCTCGAGGCCTGCTCCACTGTCCTGCCTCTCTACGCCAAGCATTATGTGCTGTGCGTGGTGAC CATCTTCTCCATCATCCTGTTGGCCATCGTGGCCCTGTACGTGCGCATCTACTGCGTGGTCCGCTC AAGCCACGCTGACATGGCCGCCCCGCAGACGCTAGCCCTGCTCAAGACGGTCACCATCGTGCTAG GCGTCTTTATCGTCTGCTGGCTGCCCGCCTTCAGCATCCTCCTTCTGGACTATGCCTGTCCCGTCCA CTCCTGCCCGATCCTCTACAAAGCCCACTACTTTTTCGCCGTCTCCACCCTGAATTCCCTGCTCAAC CCCGTCATCTACACGTGGCGCAGCCGGGACCTGCGGCGGGAGGTGCTTCGGCCGCTGCAGTGCT GGAGGCCGGGGGTGGGGGTGCAAGGACGGAGGCGGGGGGGGACCCCGGGCCACCACCTCCTG CCACTCCGCAGCTCCAGCTCCCTGGAGAGGGGCATGCACATGCCCACGTCACCCACGTTTCTGGA GGGCAACACGGTGGTCATG(SEQIDNO:3) Firefly ATGGCCGATGCTAAGAACATTAAGAAGGGCCCTGCTCCCTTCTACCCTCTGGAGGATGGCACCGC luciferase TGGCGAGCAGCTGCACAAGGCCATGAAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACC GATGCCCACATTGAGGTGGACATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGA GGCCATGAAGAGGTACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGC AGTTCTTCATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATT TACAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGTCTAA GAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAAGATCATCATC ATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGTGACATCTCATCTGCCT CCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACAGGGACAAAACCATTGCCCTGAT CATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGCGTGGCCCTGCCTCATCGCACCGCCTGTG TGCGCTTCTCTCACGCCCGCGACCCTATTTTCGGCAACCAGATCATCCCCGACACCGCTATTCTGA GCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTC GGGTGGTGCTGATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAAT TCAGTCTGCCCTGCTGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTA CGACCTGTCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAA GCCGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAACCA GCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGTGGTGCCATT CTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTGAACCAGCGCGGCGA GCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAACCCTGAGGCCACAAACGCCC TGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATTGCCTACTGGGACGAGGACGAGCACTT CTTCATCGTGGACCGCCTGAAGTCTCTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGC TGGAGTCTATCCTGCTGCAGCACCCTAACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGAC GATGCCGGCGAGCTGCCTGCCGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAG GAGATCGTGGACTATGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTG TTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCC TGATCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGTAA(SEQIDNO:4) nGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGC GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAAC ATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGGGAGATCCAAAAAAGAAGAGAAAGGTAGGCGATCCAAAAAAGAAGAGAAAG GTAGGTGATCCAAAAAAGAAGAGAAAGGTATAA(SEQIDNO:5) ASAH2 ATGAACTGCTGCATCGGGCTGGGAGAGAAAGCTCGCGGGTCCCACCGGGCCTCCTACCCAAGTC transcript TCAGCGCGCTTTTCACCGAGGCCTCAATTCTGGGATTTGGCAGCTTTGCTGTGAAAGCCCAATGG variant2 ACAGAGGACTGCAGAAAATCAACCTATCCTCCTTCAGGACCAACGTACAGAGGTGCAGTTCCATG (ACv2) GTACACCATAAATCTTGACTTACCACCCTACAAAAGATGGCATGAATTGATGCTTGACAAGGCAC CAGTGCTAAAGGTTATAGTGAATTCTCTGAAGAATATGATAAATACATTCGTGCCAAGTGGAAAA ATTATGCAGGTGGTGGATGAAAAATTGCCTGGCCTACTTGGCAACTTTCCTGGCCCTTTTGAAGA GGAAATGAAGGGTATTGCCGCTGTTACTGATATACCTTTAGGAGAGATTATTTCATTCAATATTTT TTATGAATTATTTACCATTTGTACTTCAATAGTAGCAGAAGACAAAAAAGGTCATCTAATACATGG GAGAAACATGGATTTTGGAGTATTTCTTGGGTGGAACATAAATAATGATACCTGGGTCATAACTG AGCAACTAAAACCTTTAACAGTGAATTTGGATTTCCAAAGAAACAACAAAACTGTCTTCAAGGCTT CAAGCTTTGCTGGCTATGTGGGCATGTTAACAGGATTCAAACCAGGACTGTTCAGTCTTACACTG AATGAACGTTTCAGTATAAATGGTGGTTATCTGGGTATTCTAGAATGGATTCTGGGAAAGAAAGA TGTCATGTGGATAGGGTTCCTCACTAGAACAGTTCTGGAAAATAGCACAAGTTATGAAGAAGCCA AGAATTTATTGACCAAGACCAAGATATTGGCCCCAGCCTACTTTATCCTGGGAGGCAACCAGTCT GGGGAAGGTTGTGTGATTACACGAGACAGAAAGGAATCATTGGATGTATATGAACTCGATGCTA AGCAGGGTAGATGGTATGTGGTACAAACAAATTATGACCGTTGGAAACATCCCTTCTTCCTTGAT GATCGCAGAACGCCTGCAAAGATGTGTCTGAACCGCACCAGCCAAGAGAATATCTCATTTGAAAC CATGTATGATGTCCTGTCAACAAAACCTGTCCTCAACAAGCTGACCGTATACACAACCTTGATAGA TGTTACCAAAGGTCAATTCGAAACTTACCTGCGGGACTGCCCTGACCCTTGTATAGGTTGGTGA (SEQIDNO:6) ASAH1 ATGAACTGCTGCATCGGGCTGGGAGAGAAAGCTCGCGGGTCCCACCGGGCCTCCTACCCAAGTC transcript TCAGCGCGCTTTTCACCGAGGCCTCAATTCTGGGATTTGGCAGCTTTGCTGTGAAAGCCCAATGG variant3 ACAGAGGACTGCAGAAAATCAACCTATCCTCCTTCAGGACCAACTGTCTTCCCTGCTGTTATAAGG TACAGAGGTGCAGTTCCATGGTACACCATAAATCTTGACTTACCACCCTACAAAAGATGGCATGA ATTGATGCTTGACAAGGCACCAGTGCCTGGCCTACTTGGCAACTTTCCTGGCCCTTTTGAAGAGG AAATGAAGGGTATTGCCGCTGTTACTGATATACCTTTAGGAGAGATTATTTCATTCAATATTTTTT ATGAATTATTTACCATTTGTACTTCAATAGTAGCAGAAGACAAAAAAGGTCATCTAATACATGGG AGAAACATGGATTTTGGAGTATTTCTTGGGTGGAACATAAATAATGATACCTGGGTCATAACTGA GCAACTAAAACCTTTAACAGTGAATTTGGATTTCCAAAGAAACAACAAAACTGTCTTCAAGGCTTC AAGCTTTGCTGGCTATGTGGGCATGTTAACAGGATTCAAACCAGGACTGTTCAGTCTTACACTGA ATGAACGTTTCAGTATAAATGGTGGTTATCTGGGTATTCTAGAATGGATTCTGGGAAAGAAAGAT GTCATGTGGATAGGGTTCCTCACTAGAACAGTTCTGGAAAATAGCACAAGTTATGAAGAAGCCA AGAATTTATTGACCAAGACCAAGATATTGGCCCCAGCCTACTTTATCCTGGGAGGCAACCAGTCT GGGGAAGGTTGTGTGATTACACGAGACAGAAAGGAATCATTGGATGTATATGAACTCGATGCTA AGCAGGGTAGATGGTATGTGGTACAAACAAATTATGACCGTTGGAAACATCCCTTCTTCCTTGAT GATCGCAGAACGCCTGCAAAGATGTGTCTGAACCGCACCAGCCAAGAGAATATCTCATTTGAAAC CATGTATGATGTCCTGTCAACAAAACCTGTCCTCAACAAGCTGACCGTATACACAACCTTGATAGA TGTTACCAAAGGTCAATTCGAAACTTACCTGCGGGACTGCCCTGACCCTTGTATAGGTTGGTGA (SEQIDNO:7) ASAH2 ATGGCCAAACGCACCTTCTCTAACTTGGAGACATTCCTGATTTTCCTCCTTGTAATGATGAGTGCC transcript ATCACAGTGGCCCTTCTCAGCCTCTTGTTTATCACCAGTGGGACCATTGAAAACCACAAAGATTTA variant1 GGAGGCCATTTTTTTTCAACCACCCAAAGCCCTCCAGCCACCCAGGGCTCCACAGCTGCCCAACGC TCCACAGCCACCCAGCATTCCACAGCCACCCAGAGCTCCACAGCCACTCAAACTTCTCCAGTGCCT TTAACCCCAGAGTCTCCTCTATTTCAGAACTTCAGTGGCTACCATATTGGTGTTGGACGAGCTGAC TGCACAGGACAAGTAGCAGATATCAATTTGATGGGCTATGGCAAATCCGGCCAGAATGCACAGG GCATCCTCACCAGGCTATACAGTCGTGCCTTCATCATGGCAGAACCTGATGGGTCCAATCGAACA GTGTTTGTCAGCATCGACATAGGCATGGTATCACAAAGGCTCAGGCTGGAGGTCCTGAACAGAC TGCAGAGTAAATATGGCTCCCTGTACAGAAGAGATAATGTCATCCTGAGTGGCACTCACACTCAT TCAGGTCCTGCAGGATATTTCCAGTATACCGTGTTTGTAATTGCCAGTGAAGGATTTAGCAATCAA ACTTTTCAGCACATGGTCACTGGTATCTTGAAGAGCATTGACATAGCACACACAAATATGAAACC AGGCAAAATCTTCATCAATAAAGGAAATGTGGATGGTGTGCAGATCAACAGAAGTCCGTATTCTT ACCTTCAAAATCCGCAGTCAGAGAGAGCAAGGTATTCTTCAAATACAGACAAGGAAATGATAGTT TTGAAAATGGTAGATTTGAATGGAGATGACTTGGGCCTTATCAGCTGGTTTGCCATCCACCCGGT CAGCATGAACAACAGTAACCATCTTGTAAACAGTGACAATGTGGGCTATGCATCTTACCTGCTTG AGCAAGAGAAGAACAAAGGATATCTACCTGGACAGGGGCCATTTGTAGCAGCCTTTGCTTCATCA AACCTAGGAGATGTGTCCCCCAACATTCTTGGACCACGTTGCATCAACACAGGAGAGTCCTGTGA TAACGCCAATAGCACTTGTCCCATTGGTGGGCCTAGCATGTGCATTGCTAAGGGACCTGGACAGG ATATGTTTGACAGCACACAAATTATAGGACGGGCCATGTATCAGAGAGCAAAGGAACTCTATGCC TCTGCCTCCCAGGAGGTAACAGGACCACTGGCTTCAGCACACCAGTGGGTGGATATGACAGATG TGACTGTCTGGCTCAATTCCACACATGCATCAAAAACATGTAAACCAGCATTGGGCTACAGTTTTG CAGCTGGCACTATTGATGGAGTTGGAGGCCTCAATTTTACACAGGGGAAAACAGAAGGGGATCC ATTTTGGGACACCATTCGGGACCAGATCCTGGGAAAGCCATCTGAAGAAATTAAAGAATGTCATA AACCAAAGCCCATCCTTCTTCACACCGGAGAACTATCAAAACCTCACCCCTGGCATCCAGACATTG TTGATGTTCAGATTATTACCCTTGGGTCCTTGGCCATAACTGCCATCCCCGGGGAGTTTACGACCA TGTCTGGACGAAGACTTCGAGAGGCAGTTCAAGCAGAATTTGCATCTCATGGGATGCAGAACAT GACTGTTGTTATTTCAGGTCTATGCAACGTCTATACACATTACATTACCACTTATGAAGAATACCA GGCTCAGCGATATGAGGCAGCATCGACAATTTATGGACCGCACACATTATCTGCTTACATTCAGC TCTTCAGAAACCTTGCTAAGGCTATTGCTACGGACACGGTAGCCAACCTGAGCAGAGGTCCAGAA CCTCCCTTTTTCAAACAATTAATAGTTCCATTAATTCCTAGTATTGTGGATAGAGCACCAAAAGGC AGAACTTTCGGGGATGTCCTGCAGCCAGCAAAACCTGAATACAGAGTGGGGGAAGTTGCTGAAG TTATATTTGTAGGTGCTAACCCGAAGAATTCAGTACAAAACCAGACCCATCAGACCTTCCTCACTG TGGAGAAATATGAGGCTACTTCAACATCGTGGCAGATAGTGTGTAATGATGCCTCCTGGGAGACT CGTTTTTATTGGCACAAGGGACTCCTGGGTCTGAGTAATGCAACAGTGGAATGGCATATTCCAGA CACTGCCCAGCCTGGAATCTACAGAATAAGATATTTTGGACACAATCGGAAGCAGGACATTCTGA AGCCTGCTGTCATACTTTCATTTGAAGGCACTTCCCCGGCTTTTGAAGTTGTAACTATTTAGTGA (SEQIDNO:8) ASAH2 ATGGCCAAACGCACCTTCTCTAACTTGGAGACATTCCTGATTTTCCTCCTTGTAATGATGAGTGCC transcript ATCACAGTGGCCCTTCTCAGCCTCTTGTTTATCACCAGTGGGACCATTGAAAACCACAAAGATTTA variant2 GGAGGCCATTTTTTTTCAACCACCCAAAGCCCTCCAGCCACCCAGGGCTCCACAGCTGCCCAACGC TCCACAGCCACCCAGCATTCCACAGCCACCCAGAGCTCCACAGCCACTCAAACTTCTCCAGTGCCT TTAACCCCAGAGTCTCCTCTATTTCAGAACTTCAGTGGCTACCATATTGGTGTTGGACGAGCTGAC TGCACAGGACAAGTAGCAGATATCAATTTGATGGGCTATGGCAAATCCGGCCAGAATGCACAGG GCATCCTCACCAGGCTATACAGTCGTGCCTTCATCATGGCAGAACCTGATGGGTCCAATCGAACA GTGTTTGTCAGCATCGACATAGGCATGGTATCACAAAGGCTCAGGCTGGAGGTCCTGAACAGAC TGCAGAGTAAATATGGCTCCCTGTACAGAAGAGATAATGTCATCCTGAGTGGCACTCACACTCAT TCAGGTCCTGCAGGATATTTCCAGTATACCGTGTTTGTAATTGCCAGTGAAGGATTTAGCAATCAA ACTTTTCAGCACATGGTCACTGGTATCTTGAAGAGCATTGACATAGCACACACAAATATGAAACC AGGCAAAATCTTCATCAATAAAGGAAATGTGGATGGTGTGCAGATCAACAGAAGTCCGTATTCTT ACCTTCAAAATCCGCAGTCAGAGAGAGCAAGGTATTCTTCAAATACAGACAAGGAAATGATAGTT TTGAAAATGGTAGATTTGAATGGAGATGACTTGGGCCTTATCAGCTGGTTTGCCATCCACCCGGT CAGCATGAACAACAGTAACCATCTTGTAAACAGTGACAATGTGGGCTATGCATCTTACCTGCTTG AGCAAGAGAAGAACAAAGGATATCTACCTGGACAGGGGCCATTTGTAGCAGCCTTTGCTTCATCA AACCTAGGAGATGTGTCCCCCAACATTCTTGGACCACGTTGCATCAACACAGGAGAGTCCTGTGA TAACGCCAATAGCACTTGTCCCATTGGTGGGCCTAGCATGTGCATTGCTAAGGGACCTGGACAGG ATATGTTTGACAGCACACAAATTATAGGACGGGCCATGTATCAGAGAGCAAAGTCAAAAACATGT AAACCAGCATTGGGCTACAGTTTTGCAGCTGGCACTATTGATGGAGTTGGAGGCCTCAATTTTAC ACAGGGGAAAACAGAAGGGGATCCATTTTGGGACACCATTCGGGACCAGATCCTGGGAAAGCC ATCTGAAGAAATTAAAGAATGTCATAAACCAAAGCCCATCCTTCTTCACACCGGAGAACTATCAA AACCTCACCCCTGGCATCCAGACATTGTTGATGTTCAGATTATTACCCTTGGGTCCTTGGCCATAA CTGCCATCCCCGGGGAGTTTACGACCATGTCTGGACGAAGACTTCGAGAGGCAGTTCAAGCAGA ATTTGCATCTCATGGGATGCAGAACATGACTGTTGTTATTTCAGGTCTATGCAACGTCTATACACA TTACATTACCACTTATGAAGAATACCAGGCTCAGCGATATGAGGCAGCATCGACAATTTATGGAC CGCACACATTATCTGCTTACATTCAGCTCTTCAGAAACCTTGCTAAGGCTATTGCTACGGACACGG TAGCCAACCTGAGCAGAGGTCCAGAACCTCCCTTTTTCAAACAATTAATAGTTCCATTAATTCCTA GTATTGTGGATAGAGCACCAAAAGGCAGAACTTTCGGGGATGTCCTGCAGCCAGCAAAACCTGA ATACAGAGTGGGGGAAGTTGCTGAAGTTATATTTGTAGGTGCTAACCCGAAGAATTCAGTACAA AACCAGACCCATCAGACCTTCCTCACTGTGGAGAAATATGAGGCTACTTCAACATCGTGGCAGAT AGTGTGTAATGATGCCTCCTGGGAGACTCGTTTTTATTGGCACAAGGGACTCCTGGGTCTGAGTA ATGCAACAGTGGAATGGCATATTCCAGACACTGCCCAGCCTGGAATCTACAGAATAAGATATTTT GGACACAATCGGAAGCAGGACATTCTGAAGCCTGCTGTCATACTTTCATTTGAAGGCACTTCCCC GGCTTTTGAAGTTGTAACTATTTAGTGA(SEQIDNO:9) ASAH2B ATGAGGCAGCATCGACAATTTATGGACCGCACGCATTATCTGCTTACATTCAGCTCTTCAGAAACC transcript TTGCTAAGGCTATTGCTACGTATTGTGGATAGAGCACCAAAAGGCAGAACTTTCGGGGATGTCCT variant1 GCAGCCAGCAAAACCTGAATACAGAGTGGGGGAAGTTGCTGAAGTTATATTTGTAGGTGCTAAC CCGAAGAATTCAGTACAAAACCAGACCCATCAGACCTTCCTCACTGTGGAGAAATATGAGGCTAC TTCAACATCGTGGCAGATAGTGTGTAATGATGCCTCCTGGGAGACTCGTTTTTATTGGCACAAGG GACTCCTGGGTCTGAGTAATGCAACAGTGGAATGGCATATTCCAGACACTGCCCAGCCTGGAATC TACAGAATAAGATATTTTGGACACAATCGGAAGCAGGACATTCTGAAGCCTGCTGTCATACTTTC ATTTGAAGGCACTTCCCCGGCTTTTGAAGTTGTAACTATTTAGTGA(SEQIDNO:10) ASAH2B ATGGTAGCCAACCTGAGCAGAGGTCCAGAACCTCCCTTTTTCAAACAATTAATAGTTCCATTAATT transcript CCTAGTATTGTGGATAGAGCACCAAAAGGCAGAACTTTCGGGGATGTCCTGCAGCCAGCAAAAC variant3 CTGAATACAGAGTGGGGGAAGTTGCTGAAGTTATATTTGTAGGTGCTAACCCGAAGAATTCAGT ACAAAACCAGACCCATCAGACCTTCCTCACTGTGGAGAAATATGAGGCTACTTCAACATCGTGGC AGATAGTGTGTAATGATGCCTCCTGGGAGACTCGTTTTTATTGGCACAAGGGACTCCTGGGTCTG AGTAATGCAACAGTGGAATGGCATATTCCAGACACTGCCCAGCCTGGAATCTACAGAATAAGATA TTTTGGACACAATCGGAAGCAGGACATTCTGAAGCCTGCTGTCATACTTTCATTTGAAGGCACTTC CCCGGCTTTTGAAGTTGTAACTATTTAGTGAATGGTAGCCAACCTGAGCAGAGGTCCAGAACCTC CCTTTTTCAAACAATTAATAGTTCCATTAATTCCTAGTATTGTGGATAGAGCACCAAAAGGCAGAA CTTTCGGGGATGTCCTGCAGCCAGCAAAACCTGAATACAGAGTGGGGGAAGTTGCTGAAGTTAT ATTTGTAGGTGCTAACCCGAAGAATTCAGTACAAAACCAGACCCATCAGACCTTCCTCACTGTGG AGAAATATGAGGCTACTTCAACATCGTGGCAGATAGTGTGTAATGATGCCTCCTGGGAGACTCGT TTTTATTGGCACAAGGGACTCCTGGGTCTGAGTAATGCAACAGTGGAATGGCATATTCCAGACAC TGCCCAGCCTGGAATCTACAGAATAAGATATTTTGGACACAATCGGAAGCAGGACATTCTGAAGC CTGCTGTCATACTTTCATTTGAAGGCACTTCCCCGGCTTTTGAAGTTGTAACTATTTAGTGA(SEQ IDNO:11) ASAH2B ATGGTAGCCAACCTGAGCAGAGGTCCAGAACCTCCCTTTTTCAAACAATTAATAGTTCCATTAATT transcript CCTAGTATTGTGGATAGAGCACCAAAAGGCAGAACTTTCGGGGATGTCCTGCAGCCAGCAAAAC variant4 CTGAATACAGAGTGGGGGAAGTTGCTGAAGTTATATTTGTAGGTGCTAACCCGAAGAATTCAGT ACAAAACCAGACCCATCAGACCTTCCTCACTGTGGAGAAATATGAGGCTACTTCAACATCGTGGC AGATAGTGTGTAATGATGCCTCCTGGGAGACTCGTTTTTATTGGCACAAGGGACTCCTGGGTCTG AGTAATGCAACAGTGGAATGGCATATTCCAGACACTGCCCAGCCTGGAATCTACAGAATAAGATA TTTTGGACACAATCGGAAGCAGGACATTCTGAAGCCTGCTGTCATACTTTCATTTGAAGGCACTTC CCCGGCTTTTGAAGTTGTAACTATTTAG(SEQIDNO:12) ACER1 ATGCCTAGCATCTTCGCCTATCAGAGCTCCGAGGTGGACTGGTGTGAGAGCAACTTCCAGTACTC GGAGCTGGTGGCCGAGTTCTACAACACGTTCTCCAATATCCCCTTCTTCATCTTCGGGCCACTGAT GATGCTCCTGATGCACCCGTATGCCCAGAAGCGCTCCCGCTACATTTACGTTGTCTGGGTCCTCTT CATGATCATAGGCCTGTTCTCCATGTATTTCCACATGACGCTCAGCTTCCTGGGCCAGCTGCTGGA CGAGATCGCCATCCTGTGGCTCCTGGGCAGTGGCTATAGCATATGGATGCCCCGCTGCTATTTCC CCTCCTTCCTTGGGGGGAACAGGTCCCAGTTCATCCGCCTGGTCTTCATCACCACTGTGGTCAGCA CCCTTCTGTCCTTCCTGCGGCCCACGGTCAACGCCTACGCCCTCAACAGCATTGCCCTGCACATTCT CTACATCGTGTGCCAGGAGTACAGGAAGACCAGCAATAAGGAGCTTCGGCACCTGATTGAGGTC TCCGTGGTTTTATGGGCTGTTGCTCTGACCAGCTGGATCAGTGACCGTCTGCTTTGCAGCTTCTGG CAGAGGATTCATTTCTTCTATCTGCACAGCATCTGGCATGTGCTCATCAGCATCACCTTCCCTTATG GCATGGTCACCATGGCCTTGGTGGATGCCAACTATGAGATGCCAGGTGAAACCCTCAAAGTCCGC TACTGGCCTCGGGACAGTTGGCCCGTGGGGCTGCCCTACGTGGAAATCCGGGGTGATGACAAGG ACTGCTGA(SEQIDNO:13) ACER2 ATGGGCGCCCCGCACTGGTGGGACCAGCTGCAGGCTGGTAGCTCGGAGGTGGACTGGTGCGAG GACAACTACACCATCGTGCCTGCTATCGCCGAGTTCTACAACACGATCAGCAATGTCTTATTTTTC ATTTTACCGCCCATCTGCATGTGCTTGTTTCGTCAGTATGCAACATGCTTCAACAGTGGCATCTACT TAATCTGGACTCTTTTGGTTGTAGTGGGAATTGGATCCGTCTACTTCCATGCAACCCTTAGTTTCTT GGGTCAGATGCTTGATGAACTTGCAGTCCTTTGGGTTCTGATGTGTGCTTTGGCCATGTGGTTCCC CAGAAGGTATCTACCAAAGATCTTTCGGAATGACCGGGGTAGGTTCAAGGTGGTGGTCAGTGTC CTGTCTGCGGTTACGACGTGCCTGGCATTTGTCAAGCCTGCCATCAACAACATCTCTCTGATGACC CTGGGAGTTCCTTGCACTGCACTGCTCATCGCAGAGCTAAAGAGGTGTGACAACATGCGTGTGTT TAAGCTGGGCCTCTTCTCGGGCCTCTGGTGGACCCTGGCCCTGTTCTGCTGGATCAGTGACCGAG CTTTCTGCGAGCTGCTGTCATCCTTCAACTTCCCCTACCTGCACTGCATGTGGCACATCCTCATCTG CCTTGCTGCCTACCTGGGCTGTGTATGCTTTGCCTACTTTGATGCTGCCTCAGAGATTCCTGAGCA AGGCCCTGTCATCAAGTTCTGGCCCAATGAGAAATGGGCCTTCATTGGTGTCCCCTATGTGTCCCT CCTGTGTGCCAACAAGAAATCATCAGTCAAGATCACGTGA(SEQIDNO:14) ACER3 ATGGCTCCGGCCGCGGACCGAGAGGGCTACTGGGGCCCCACGACCTCCACGCTGGACTGGTGCG transcript AGGAGAACTACTCCGTGACCTGGTACATCGCCGAGTTCTGGAATACAGTGAGTAACCTGATCATG variant1 ATTATACCTCCAATGTTCGGTGCAGTTCAGAGTGTTAGAGACGGTCTGGAAAAGCGGTACATTGC TTCTTATTTAGCACTCACAGTGGTAGGAATGGGATCCTGGTGCTTCCACATGACTCTGAAATATGA AATGCAGCTATTGGATGAACTCCCAATGATATACAGCTGTTGCATATTTGTGTACTGCATGTTTGA ATGTTTCAAGATCAAGAACTCAGTAAACTACCATCTGCTTTTTACCTTAGTTCTATTCAGTTTAATA GTAACCACAGTTTACCTTAAGGTAAAAGAGCCGATATTCCATCAGGTCATGTATGGAATGTTGGT CTTTACATTAGTACTTCGATCTATTTATATTGTTACATGGGTTTATCCATGGCTTAGAGGACTGGGT TATACATCATTGGGTATATTTTTATTGGGATTTTTATTTTGGAATATAGATAACATATTTTGTGAGT CACTGAGGAACTTTCGAAAGAAGGTACCACCTATCATAGGTATTACCACACAATTTCATGCATGG TGGCATATTTTAACTGGCCTTGGTTCCTATCTTCACATCCTTTTCAGTTTGTATACAAGAACACTTT ACCTGAGATATAGGCCAAAAGTGAAGTTTCTCTTTGGAATCTGGCCAGTGATCCTGTTTGAGCCTC TCAGGAAGCATTGA(SEQIDNO:15) ACER3 ATGGCTCCGGCCGCGGACCGAGAGGGCTACTGGGGCCCCACGACCTCCACGCTGGACTGGTGCG transcript AGGAGAACTACTCCGTGACCTGGTACATCGCCGAGTTCTTGGTAGGAATGGGATCCTGGTGCTTC variant2 CACATGACTCTGAAATATGAAATGCAGCTATTGGATGAACTCCCAATGATATACAGCTGTTGCAT ATTTGTGTACTGCATGTTTGAATGTTTCAAGATCAAGAACTCAGTAAACTACCATCTGCTTTTTACC TTAGTTCTATTCAGTTTAATAGTAACCACAGTTTACCTTAAGGTAAAAGAGCCGATATTCCATCAG GTCATGTATGGAATGTTGGTCTTTACATTAGTACTTCGATCTATTTATATTGTTACATGGGTTTATC CATGGCTTAGAGGACTGGGTTATACATCATTGGGTATATTTTTATTGGGATTTTTATTTTGGAATA TAGATAACATATTTTGTGAGTCACTGAGGAACTTTCGAAAGAAGGTACCACCTATCATAGGTATT ACCACACAATTTCATGCATGGTGGCATATTTTAACTGGCCTTGGTTCCTATCTTCACATCCTTTTCA GTTTGTATACAAGAACACTTTACCTGAGATATAGGCCAAAAGTGAAGTTTCTCTTTGGAATCTGGC CAGTGATCCTGTTTGAGCCTCTCAGGAAGCATTGA(SEQIDNO:16) ACER3 ATGATATACAGCTGTTGCATATTTGTGTACTGCATGTTTGAATGTTTCAAGATCAAGAACTCAGTA transcript AACTACCATCTGCTTTTTACCTTAGTTCTATTCAGTTTAATAGTAACCACAGTTTACCTTAAGGTAA variant3 AAGAGCCGATATTCCATCAGGTCATGTATGGAATGTTGGTCTTTACATTAGTACTTCGATCTATTT ATATTGTTACATGGGTTTATCCATGGCTTAGAGGACTGGGTTATACATCATTGGGTATATTTTTAT TGGGATTTTTATTTTGGAATATAGATAACATATTTTGTGAGTCACTGAGGAACTTTCGAAAGAAG GTACCACCTATCATAGGTATTACCACACAATTTCATGCATGGTGGCATATTTTAACTGGCCTTGGT TCCTATCTTCACATCCTTTTCAGTTTGTATACAAGAACACTTTACCTGAGATATAGGCCAAAAGTGA AGTTTCTCTTTGGAATCTGGCCAGTGATCCTGTTTGAGCCTCTCAGGAAGCATIGA(SEQIDNO: 17) Sphk2 ATGAATGGACACCTTGAAGCAGAGGAGCAGCAGGACCAGAGGCCAGACCAGGAGCTGACCGGG AGCTGGGGCCACGGGCCTAGGAGCACCCTGGTCAGGGCTAAGGCCATGGCCCCGCCCCCACCGC CACTGGCTGCCAGCACCCCGCTCCTCCATGGCGAGTTTGGCTCCTACCCAGCCCGAGGCCCACGC TTTGCCCTCACCCTTACATCGCAGGCCCTGCACATACAGCGGCTGCGCCCCAAACCTGAAGCCAG GCCCCGGGGTGGCCTGGTCCCGTTGGCCGAGGTCTCAGGCTGCTGCACCCTGCGAAGCCGCAGC CCCTCAGACTCAGCGGCCTACTTCTGCATCTACACCTACCCTCGGGGCCGGCGCGGGGCCCGGCG CAGAGCCACTCGCACCTTCCGGGCAGATGGGGCCGCCACCTACGAAGAGAACCGTGCCGAGGCC CAGCGCTGGGCCACTGCCCTCACCTGTCTGCTCCGAGGACTGCCACTGCCCGGGGATGGGGAGA TCACCCCTGACCTGCTACCTCGGCCGCCCCGGTTGCTTCTATTGGTCAATCCCTTTGGGGGTCGGG GCCTGGCCTGGCAGTGGTGTAAGAACCACGTGCTTCCCATGATCTCTGAAGCTGGGCTGTCCTTC AACCTCATCCAGACAGAACGACAGAACCACGCCCGGGAGCTGGTCCAGGGGCTGAGCCTGAGTG AGTGGGATGGCATCGTCACGGTCTCGGGAGACGGGCTGCTCCATGAGGTGCTGAACGGGCTCCT AGATCGCCCTGACTGGGAGGAAGCTGTGAAGATGCCTGTGGGCATCCTCCCCTGCGGCTCGGGC AACGCGCTGGCCGGAGCAGTGAACCAGCACGGGGGATTTGAGCCAGCCCTGGGCCTCGACCTGT TGCTCAACTGCTCACTGTTGCTGTGCCGGGGTGGTGGCCACCCACTGGACCTGCTCTCCGTGACG CTGGCCTCGGGCTCCCGCTGTTTCTCCTTCCTGTCTGTGGCCTGGGGCTTCGTGTCAGATGTGGAT ATCCAGAGCGAGCGCTTCAGGGCCTTGGGCAGTGCCCGCTTCACACTGGGCACGGTGCTGGGCC TCGCCACACTGCACACCTACCGCGGACGCCTCTCCTACCTCCCCGCCACTGTGGAACCTGCCTCGC CCACCCCTGCCCATAGCCTGCCTCGTGCCAAGTCGGAGCTGACCCTAACCCCAGACCCAGCCCCG CCCATGGCCCACTCACCCCTGCATCGTTCTGTGTCTGACCTGCCTCTTCCCCTGCCCCAGCCTGCCC TGGCCTCTCCTGGCTCGCCAGAACCCCTGCCCATCCTGTCCCTCAACGGTGGGGGCCCAGAGCTG GCTGGGGACTGGGGTGGGGCTGGGGATGCTCCGCTGTCCCCGGACCCACTGCTGTCTTCACCTC CTGGCTCTCCCAAGGCAGCTCTACACTCACCCGTCTCCGAAGGGGCCCCCGTAATTCCCCCATCCT CTGGGCTCCCACTTCCCACCCCTGATGCCCGGGTAGGGGCCTCCACCTGCGGCCCGCCCGACCAC CTGCTGCCTCCGCTGGGCACCCCGCTGCCCCCAGACTGGGTGACGCTGGAGGGGGACTTTGTGC TCATGTTGGCCATCTCGCCCAGCCACCTAGGCGCTGACCTGGTGGCAGCTCCGCATGCGCGCTTC GACGACGGCCTGGTGCACCTGTGCTGGGTGCGTAGCGGCATCTCGCGGGCTGCGCTGCTGCGCC TTTTCTTGGCCATGGAGCGTGGTAGCCACTTCAGCCTGGGCTGTCCGCAGCTGGGCTACGCCGCG GCCCGTGCCTTCCGCCTAGAGCCGCTCACACCACGCGGCGTGCTCACAGTGGACGGGGAGCAGG TGGAGTATGGGCCGCTACAGGCACAGATGCACCCTGGCATCGGTACACTGCTCACTGGGCCTCCT GGCTGCCCGGGGGGGGAGCCCTGA(SEQIDNO:18) CerK ATGGGGGCGACGGGGGCGGCGGAGCCGCTGCAATCCGTGCTGTGGGTGAAGCAGCAGCGCTGC GCCGTGAGCCTGGAGCCCGCGCGGGCTCTGCTGCGCTGGTGGCGGAGCCCGGGGCCCGGAGCC GGCGCCCCCGGCGCGGATGCCTGCTCTGTGCCTGTATCTGAGATCATCGCCGTTGAGGAAACAG ACGTTCACGGGAAACATCAAGGCAGTGGAAAATGGCAGAAAATGGAAAAGCCTTACGCTTTTAC AGTTCACTGTGTAAAGAGAGCACGACGGCACCGCTGGAAGTGGGCGCAGGTGACTTTCTGGTGT CCAGAGGAGCAGCTGTGTCACTTGTGGCTGCAGACCCTGCGGGAGATGCTGGAGAAGCTGACGT CCAGACCAAAGCATTTACTGGTATTTATCAACCCGTTTGGAGGAAAAGGACAAGGCAAGCGGAT ATATGAAAGAAAAGTGGCACCACTGTTCACCTTAGCCTCCATCACCACTGACATCATCGTTACTGA ACATGCTAATCAGGCCAAGGAGACTCTGTATGAGATTAACATAGACAAATACGACGGCATCGTCT GTGTCGGCGGAGATGGTATGTTCAGCGAGGTGCTGCACGGTCTGATTGGGAGGACGCAGAGGA GCGCCGGGGTCGACCAGAACCACCCCCGGGCTGTGCTGGTCCCCAGTAGCCTCCGGATTGGAAT CATTCCCGCAGGGTCAACGGACTGCGTGTGTTACTCCACCGTGGGCACCAGCGACGCAGAAACCT CGGCGCTGCATATCGTTGTTGGGGACTCGCTGGCCATGGATGTGTCCTCAGTCCACCACAACAGC ACACTCCTTCGCTACTCCGTGTCCCTGCTGGGCTACGGCTTCTACGGGGACATCATCAAGGACAG TGAGAAGAAACGGTGGTTGGGTCTTGCCAGATACGACTTTTCAGGTTTAAAGACCTTCCTCTCCC ACCACTGCTATGAAGGGACAGTGTCCTTCCTCCCTGCACAACACACGGTGGGATCTCCAAGGGAT AGGAAGCCCTGCCGGGCAGGATGCTTTGTTTGCAGGCAAAGCAAGCAGCAGCTGGAGGAGGAG CAGAAGAAAGCACTGTATGGTTTGGAAGCTGCGGAGGACGTGGAGGAGTGGCAAGTCGTCTGT GGGAAGTTTCTGGCCATCAATGCCACAAACATGTCCTGTGCTTGTCGCCGGAGCCCCAGGGGCCT CTCCCCGGCTGCCCACTTGGGAGACGGGTCTTCTGACCTCATCCTCATCCGGAAATGCTCCAGGTT CAATTTTCTGAGATTTCTCATCAGGCACACCAACCAGCAGGACCAGTTTGACTTCACTTTTGTTGA AGTTTATCGCGTCAAGAAATTCCAGTTTACGTCGAAGCACATGGAGGATGAGGACAGCGACCTC AAGGAGGGGGGGAAGAAGCGCTTTGGGCACATTTGCAGCAGCCACCCCTCCTGCTGCTGCACCG TCTCCAACAGCTCCTGGAACTGCGACGGGGAGGTCCTGCACAGCCCTGCCATCGAGGTCAGAGT CCACTGCCAGCTGGTTCGACTCTTTGCACGAGGAATTGAAGAGAATCCGAAGCCAGACTCACACA GCTGA (SEQIDNO:19)
Reducing Cell Death in Rat Myocardium

(48) In order to characterize the dynamics of cell death as well as expression of genes that are involved in the metabolism and signaling of sphingolipids in the heart as a result of myocardial infarction (MI) in mice, hearts were infarcted by ligation of the left anterior descending artery (LAD) and harvested at different time points post ligation.

(49) For cell death assessment the hearts were harvested at 1, 2, 4, and 28 days post MI and from sham operated mice. TUNEL stain was used to assess DNA fragmentation in cardiac cells. Troponin-I immunostaining was used to distinguish between cardiomyocytes and non-cardiomyocytes (FIG. 1A). The highest level of DNA fragmentation was found 24 h post MI with 92% of total cells in LV having fragmented DNA, 153% of CM and 40.2% of non CM. The levels of DNA fragmentation two days post MI reduced both in CM and non CM and reached to basal levels 28 d post MI with 0.10.1% of total cells 0.070.08% of CM and 0.120.1% of non CM comparable to the levels in the hearts of control mice. Cleaved Caspase3 immunoblotting 24 h post MI confirmed high levels of cell death in the infarcted area (FIG. 5C).

(50) Sphingolipid metabolism and signaling pathway partial transcriptomes were studied in hearts of sham operated mice or mice 4 h and 24 h post MI. We focused on two partially overlapping sets of genes: Sphingolipid metabolism genes based on KEGG PATHWAY map00600 and Sphingolipid signaling pathway genes based on KEGG PATHWAY map04071 [11]. In the Sphingolipid metabolism transcriptome 4 h post ligation, 2 genes were significantly upregulated by more than 2 fold and one was downregulated by less than 2 fold. 24 h post MI, 10 genes were significantly upregulated by more than 2 fold and 2 were downregulated by less than 2 fold. A total of 12 out of 39 genes (not shown) related to sphingolipid metabolism were significantly upregulated. In the Sphingolipids signaling pathway transcriptome 4 h post ligation 5 genes were significantly upregulated by more than 2 fold and 2 were downregulated by less than 2 fold. 24 h post MI, 28 genes were significantly upregulated by more than 2 fold and 10 were downregulated by less than 2 fold totals of 38 out of 82 genes (FIG. 1B and FIG. 5)

(51) The dendrograms of both transcriptomes (FIG. 1B and FIG. 5A) shows that the control group and the 4 h post MI group are clustered together while the 24 h post MI group is clustered as a separate group suggesting that the major alterations in sphingolipids metabolism and signaling pathway related genes expression occurs more than 4 h post MI.

(52) In order to study the role of ceramide metabolites on cell death and heart function post MI we chose to alter ceramide metabolism and signaling pathway by enhancing ceramide hydrolysis and S1P formation. First we confirm the RNA-seq DATA for the main genes that are involved in this process namely: Acid ceramidase (AC), Sphingosine Kinase 1 (Sphk1) and Sphingosine-1-Phosphate Receptor 2 (S1PR2) by qPCR and western blot analysis of hearts from an independent experiment. In agreement with the results of the RNAseq analysis, the relative levels of AC mRNA didn't change significantly (FIG. 1B). The levels of AC precursor did not change however, the levels of AC subunit and subunit gradually increased during infarct development (FIG. 1C) The increase in and subunits is accompanied by an increase in the activity level of AC (FIG. 1D). The mRNA levels of Sphk1 increased by 6 times at 4 h and by 35 times at 24 h. Western blot analysis revealed a dramatic increase in the levels of Sphk1 protein 4 h and 24 h post MI (FIGS. 1B and C and FIG. 5D). The relative levels of S1PR2 mRNA declined by 50% 4 h post MI and returned to normal after 24 h. The levels of S1PR2 did not change 4 h or 24 h post MI (FIG. 1B and FIG. 5E).

(53) Acid ceramidase catalyzes the hydrolysis of ceramide into sphingosine and free fatty acid [18]. While it has been reported that sphingosine is capable of disassembling mitochondrial ceramide channels suggesting the existence of an anti-apoptotic property of sphingosine [19, 20] other evidence support a positive role of sphingosine in the execution of apoptotic or necrotic cell death [21]. Moreover, it was suggested by Benaim et al [22] that sphingosine can disturb the homeostasis of cellular calcium by inhibiting the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) which has a pivotal role in proper cardiac function [23, 24]. The two genes that encode sphingosine kinase, Sphk1 and Sphk2, catalyze the phosphorylation of sphingosine to S1P and have shown to possess cardioprotective properties [25]. Duan et al reported that adenoviral mediated overexpression of Sphk1 in rat hearts can protect the treated hearts from ischemia and reperfusion injury [26]. Our transcriptome analysis shows that the expression levels of Sphk1 are elevated by 12 and 67 fold 4 and 24 hours post-MI respectively. A similar trend was found with qPCR analyzed of Sphk1 levels in an independent experiment. This was accompanied by a significant elevation in Sphk1 protein levels as measured by western blot analysis. The pathway analysis of sphingosine signal transduction revealed up regulation of all the components in the TNF signaling pathway including TNF alpha, TNFR, TRADD, and TRAF2. Interestingly, Xia et al showed that TRAF2 can interact with Sphk1 and that this interaction is necessary for the anti-apoptotic activity of TRAF2 [27]. Recently Guo et al reported a cardioprotective role of TRAF2 [28].

(54) Sphingosine 1 phosphate exerts its activity on cells by activating a family of five G protein-coupled receptors: S1pr1-5. The levels of the two most abundant receptors in the heart namely S1pr1 and 3 are moderately but significantly elevated after MI. In contrast, the levels of S1pr2 4 h after MI are reduced and 24 h post MI the levels are back to normal. The role of S1pr1 and S1pr3 in cardio protection is well established [25] however the role of S1p2 in heart function is less clear. Or results suggest that overexpression of S1p2 in cells and in heart have a neglected effect on cells survival.

(55) Cell Senescence

(56) Senescence is the major cause of suffering, disease, and death in modern times. Senescence, or biological aging, is the slow drop of functional characteristics. Senescence can refer either to cellular senescence or to the senescence of organs or a whole organism. In addition to induced senescence such as aging, there is stress-induced senescence, which is a broad concept including a variety of stress conditions such as oxidative stress, injury, noise exposure, and other sources of damage to cells. These stresses act via intracellular pathways to a state of non-proliferation. Cellular senescence described by Hayflick and Moorhead in the 1960s, is the irreversible arrest of cells following long culture. Telomere shortening is the key mechanism driving replicative senescence in human fibroblasts. Apart from cell cycle arrest, senescent cells have been shown to experience dramatic changes in terms of gene expression, combination of CDK1 activity, heterochromatin formation, metabolism including (sphingolipids metabolism), epigenetics, and a distinct secretion profile known as the Senescence-Associated Secretory Phenotype (SASP) (Copp'e et al., 2014). Senescent cells use the SASP to communicate with the immune system, potentially to facilitate their own clearance (for example pro-inflammatory cytokines) and contribute to disruption of cell and tissue homeostasis and function (Shay and Wright, 2010). It has been shown that chronic SASP is able to induce senescence in adjacent young cells, contributing to tissue dysfunction (Acosta et al., 2013, Jurk et al., 2014). Senescent cells also show mitochondrial dysfunction (Passos et al., 2010).

(57) Oxidative stress-induced senescence in the heart caused by myocardial infarction (MI) can trigger cardiomyocyte death or senescence (Huitong et al., 2018). Moreover, senescence can have deleterious effects with chronic, worsening pathologies such as type 2 diabetes (Palmer et al., 2015), atherosclerosis (Gorenne et al., 2006; Wang et al., 2015), Multiple Sclerosis (MS) (Oost et al., 2019), and other chronic diseases.

(58) The involvement of sphingolipids has been studied in multiple organisms and cell types for the regulation of aging and senescence, especially ceramide and sphingosine-1-phosphate (S1P) for induced cellular senescence, distinct from their effect on survival. Significant and wide-ranging evidence defines critical roles of sphingolipid enzymes and pathways in aging and organ injury leading to tissue senescence (Trayssac et al., 2018), including regulation by stress stimuli, p53, participation in growth arrest, SASP, and other aspects of the senescence response. Acid ceramidase is the only protein that can balance the level of ceramide vs S1P by hydrolyzing ceramide to a product that can be phosphorylated to form S1P. The present invention is based on the further discovery that in addition to its role in protecting cells from apoptosis, administration of AC decreased the rate of senescence in vitro, and in vivo, in different cell types and tissues.

(59) Blockage in the coronary arteries reduces the supply of blood to heart muscle and causes dynamic effects within the infarction risk area and around the ischemic border zone. Tissues in the infarction risk area exhibit distinct metabolic changes within a few minutes. Nearly the entire risk area tissues become irreversibly injured during a severe hypoperfusion of 6 hours. On the other hand, the border zone tissues exhibit only moderate metabolic changes due to greater collateral perfusion, including from 45-80% of blood flow regionally in the non-ischemic vascular bed. The ischemic border zone tissues are from the lateral edges of infarct, are approximately 2 mm wide, and increase in width along the subepicardium. Over time, the subepicardial margins of border zone widen due to improved collateral blood flow. The tissues in the border zone region are in, or entering into, senescence.

(60) We tested the effect of AC gene therapy on induced cardiomyocyte senescence in sheep hearts post ischemic injury, using an Anc80 vector encoding AC. Proteomic analysis of the sheep hearts post ischemic injury detected expression of over 4000 genes. These were refined to 1500 genes by known senescence and age-related gene databases. Functional analysis of the heart post the ischemic stress revealed that there are changes in expression of gene related to senescence mainly in the boarder zone area. Significant changes were observed to 11 out of 25 genes with known roles in the KEGG cellular senescence pathways. Treatment with AC-Anc80 post ischemic injury presented expression levels consistent with control hearts (no ischemic injury) in 10 out of the 11 detected senescence genes. For example, TXN, a major transcription factor involved in senescence and up-regulation of the p53/p21 and p16 tumor suppressor pathways, was differentially expressed. In addition, TP53BP1, a major messenger in DNA damage responses, along with TP53, ATM and other ageing-associated players, was up-regulated post ischemic injury and presented normal levels post treatment with AC-Anc80.

(61) Also, 6 of 8 detected collagen genes that are down-regulated during senescence, were highly elevated above the level of control post AC-Anc80 treatment. These results suggest that AC can be used to prevent senescence\aging in skin cells. PRELP deficiency has been reported to account for many symptoms of Hutchinson-Gilford progeria (HGP). Interestingly PRELP was highly up-regulated post AC-Anc80 treatment. We propose testing the possibility of using AC-Anc80 gene therapy for HGP disease. Moreover, inhibition of FABP4 was recently shown to induce senescence of endothelial cells such as insulin resistance, diabetes mellitus, atherosclerosis, hypertension, cardiac dysfunction (Furuhashi, et al, 2014). FABP4 was down regulated post ischemia and up regulated post AC-Anc80 treatment. Base on these results AC treatments can be applied to prevent senescence in different types of tissues composed of endothelial cells.

Examples

(62) Mice

(63) All animal procedures were performed under protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Care and Use Committee. CFW mice strains, male and female, were used for studies on heart function following myocardial infarction. Before surgery mice were anaesthetized with ketamine 100 mg/kg and xylazine 10 mg/kg cocktail.

(64) hPSC Differentiation

(65) For heart function following myocardial infarction studies, hematoPoietic Stem Cells (hPSCs) (H9) were differentiated along a cardiac lineage as previously described. Briefly, hPSCs were maintained in E8 media and passaged every 4-5 days onto matrigel-coated plates. To generate embryonic bodies (EBs), hPSCs were treated with 1 mg/ml collagenase B (Roche) for 30 min or until cells dissociated from plates. Cells were collected and centrifuged at 1,300 rpm for 3 min, and they were resuspended into small clusters of 50-100 cells by gentle pipetting in differentiation media containing RPMI (Gibco), 2 mmol/L L-glutamine (Invitrogen), 410 monothioglycerol (MTG, Sigma), 50 mg/mL ascorbic acid (Sigma), and 150 mg/mL transferrin (Roche). Differentiation media were supplemented with 2 ng/mL BMP4 and 3 mmol Thiazovivin (Millipore) (day 0). EBs were maintained in six-well ultra-low attachment plates (Corning) at 37 C. in 5% CO2, 5% O2, and 90% N2. On day 1, media were changed to differentiation media supplemented with 20 ng/mL BMP4 (R&D Systems) and 20 ng/mL Activin A (R&D Systems). On day 4, media were changed to differentiation media supplemented with 5 ng/mL VEGF (R&D Systems) and 5 mmol/L XAV (Stemgent). After day 8, media were changed every 5 days to differentiation media without supplements.

(66) Synthesis of Anc80.AC

(67) The nucleotide sequence for an embodiment of the Anc80 plasmid described herein is shown below. A map of the vector is also shown in FIG. 16.

(68) Anc80 Plasmid Sequence

(69) TABLE-US-00002 pAAV.CMV. CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG WPRE.bGH.d GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG na GAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCC ATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCGCCCTTAAG CTAGCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTC ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGG TTTAGTGAACCGTCAGATCCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGT AAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTG TCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA CATCCACTTTGCCTTTCTCTCCACAGGTGTCCAGGCGGCCGCNNNGGATCCA ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATG TTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT CTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCAC TGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAG CTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCA TCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTG ACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGC CTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCG GCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGAGATCTGCCTCGACTGTGCCTTCTAGTTGCCAGCC ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACT CCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGG ATTGGGAAGACAATAGCAGGCATGCTGGGGACTCGAGTTAAGGGCGAATTC CCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGT TAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTG CGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTA ACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC GTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGA ATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTG GTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCA AGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGC CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTT TGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA TTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGG TGGCATCTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAA TACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAAT AATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATT CCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTT TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTA TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACA GTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCA ACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAAT GAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAA CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAA CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACAT ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG GTTCCTGGCCTTTTGCTGCGGTTTTGCTCACATGTTCTTTCCTGCGTTATCCC CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCG CCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGC AATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGC TTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGG AAACAGCTATGACCATGATTACGCCAGATTTAATTAAGG(SEQIDNO:20)
Sheep Model of Myocardial Infarction and Gene Delivery with Anc80

(70) All work was approved by the IACUC at the Mount Sinai School of Medicine. Male sheep were subjected to left surgical thoracotomy to expose the heart. To induce severe myocardial infarction, two select arteries of the coronary artery tree off the main circumflex artery were ligated closed with 7.0 prolene suture to occlude blood flow. The lateral to posterolateral wall region was visibly demarcated with hypoxia indication below the ligations. Confirmation of ST segment elevation confirmed infarct for each procedure. For gene therapy, 1.5 mL of Anc80.AC gene was prepared in two 1 cc syringes with a 26 gauge needle. A total of 15 injections each containing 100 L was performed directly inside the discolored, infarcted myocardium in the upper basal and middle slices where the majority of myocardium at risk presented. The lower portion apical area was not injected. The animal was closed, recovered and sent for MRI evaluation at 4 weeks and 3 months post infarction.

(71) Total RNA was isolated using the RNeasy mini kit (QIAGEN) and reverse transcribed using Superscript III reverse transcriptase (Invitrogen), according to the manufacturer's instructions. Real-time qPCR analyses were performed on a Mastercycler realplex 4 Sequence Detector (Eppendoff) using SYBR Green (Quantitect SYBR Green PCR Kit, QIAGEN). Data were normalized to 18srRNA expression where appropriate (endogenous controls). Fold changes of gene expression were determined by the ddCT method. PCR primer sequences are summarized in Table 2.

(72) TABLE-US-00003 TABLE2 SEQ SEQ ID ID Gene Forward NO. Reverse NO. AC ACAGGATTCAAACCAGGACTGT 21 TGGGCATCTTTCCTTCCGAA 22 AC TGACAGGATTCAAACCAGGACT 23 CTGGGCATCTTTCCTTCCGA 24 Sphk1 ATACTCACCGAACGGAAGAACC 25 CCATTAGCCCATTCACCACCTC 26 Sphk1 ACTGATACTCACCGAACGGAA 27 CATTAGCCCATTCACCACCTC 28 S1PR2 CACAGCCAACAGTCTCCAAA 29 TCTGAGTATAAGCCGCCCA 30 S1PR2 ATAGACCGAGCACAGCCAA 31 GAACCTTCTCAGGATTGAGGT 32 18srRNA* TAACGAACGAGACTCTGGCAT 33 CGGACATCTAAGGGCATCACA 34 G *Genetic Vaccines and Therapy 2004, 2:5
Western Blot

(73) Upon thawing, hearts lysates' were subjected to separation by SDS-PAGE using 12% precast Nupage Bis/Tris gels (Invitrogen, Carlsbad, CA, USA) under reducing conditions and MES running buffer (Invitrogen), and transferred onto a nitrocellulose membrane (Bio-Rad) using a semidry transfer apparatus and Nupage-MOPS transfer buffer (Invitrogen). The membrane was block with TBS/Tween containing 5% dry milk and incubated with specific primary antibodies over night at 4 C. washed with TBS/Tween and incubated with rabbit or goat antibodies conjugated to horseradish peroxidase for 1 hour at room temperature. Detection was performed by an enhanced chemiluminecence (ECL) detection system (Pierce, Rockford, IL). For molecular weight determination prestained protein standards (Amersham, Buckinghamshire, UK) were used.

(74) Immunohistochemistry

(75) The mouse hearts were harvested and perfused using perfusion buffer (2 g/l butanedione, monoxime and 7.4 g/l KCl in PBSx1) and 4% paraformaldehyde (PFA). Hearts were fixed in 4% PFA/PBS overnight on shaker and then washed with PBS for 1 hr and incubated in 30% sucrose/PBS at 40 C overnight. Before freezing, hearts were mounted in OCT for 30 min and frozen at 80 C. Transverse heart sections of 10 M were made by cryostat. Cryosections were washed in PBST and blocked for 1 h with 5% donkey serum in PBST. Sections were incubated over night at 4 C. using primary antibodies for Troponin I, Sphk1, S1p2. Secondary antibodies were used for fluorescent labeling (Jackson ImmunoResearch Laboratories). TUNEL staining was performed according to manufacturer's recommendations (In-Situ Cell Death Detection Kit, Fluorescein, Cat #11684795910, Roche). Stained sections were imaged using a Zeiss Slide Scanner Axio Scan or Zeiss mic. Quantification of TUNEL in cardiac sections was performed using ImageJ software. For cell immunocytochemistry, Hek293 and isolated CMs were fixed on coverslips with 4% PFA for 10 min at room temperature. Following permeabilization with 0.1% TRITON X100 in PBS for 10 min at room temperature, cells were blocked with 5% Donkey serum+0.1% TRITON X100 in PBS for 30 minutes. Coverslips were incubated with primary antibodies in humidity chamber for 1 hour at room temperature followed by incubation with corresponding secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 647 and Alexa Fluor 555, and Hoechst 33342 staining for nuclei visualization (all from Invitrogene). The fluorescent images were taken on a Zeiss fluorescent microscope at 20 magnification.

(76) Cardiac MRI Background

(77) Cardiac magnetic resonance imaging (CMRI) is gold standard for the evaluation of volume, dimensions, structure and complete diagnostic profile of myocardium. The advantages of MRI over all other imaging modalities including echocardiography and CT are well established and are: (1) non user dependency (2) high reproducibility (3) averaging of several hundred heartbeats (4) high spatial/temporal resolution programmed without inter or intra subject variability (5) non-invasive means to analyze viability, inflammation and metabolic profile without using probes or invasive lines. CMRI is acquired over the course of a 1 hour exam inside a standard magnet with clinical protocol. In these experiments: 45 kg male sheep were intubated, maintained on anesthesia and placed into a Siemens Skyra 3T magnet. Standard long axis, short axis, and t2 weighted maps along with contrast to determine infarct size were performed on baseline and follow up studies. 15 mL of gadolinium contrast was used to assess myocardial infarction size and tissue characteristics per standard clinical protocols. An experienced, blinded user analyzed the DICOM files offline.

(78) Injection of Anc80.AC into rat hearts shows higher AC activity in the heart tissue when compared to untreated hearts (control 34 nM vs Anc80.AC 12 nM).

(79) Results in sheep demonstrate that treatment with Anc80.AC immediately after myocardial infarction (MI) leads to complete rescue of the MI injected area with very robust contractility. The heart function results are excellent, greater than 60%, which is in the range for baseline animals. Only very minor scarring was detected in the non-injected area. No effect on heart rate and no indication of myocarditis was observed.

(80) These findings suggest that by modulating ceramide levels from stressed cells and elevating S1P, the cell death pathway is inhibited and cell survival is initiated, in vivo.

(81) In vivo applications include administration of recombinant AC variant 1 (rACv1). In one embodiment, 0.005 g/l of rACV1 is admixed into a gel or cream to be administered to the skin in order to prevent cell death or reverse senescence/aging of epithelial skin cells.

(82) In another embodiment, 0.005 g/l of rACV1 is admixed into a suitable eye drop preparation in order to prevent cell death or reverse senescence/aging of cone cells post stress-related reaction.

(83) More than 90 percent of hearing loss occurs when either hair cells or auditory nerve cells are destroyed. The present method provides an opportunity to prevent or reduce the loss of hair cells, thereby reducing the likelihood of hearing loss.

(84) It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the forgoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

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