ANC80 ENCODING SPHINGOLIPID-METABOLIZING PROTEINS FOR MITIGATING DISEASE-INDUCED TISSUE DAMAGE

Abstract

The present disclosure relates generally to the use of sphingolipid-metabolizing proteins to mitigate or minimize tissue damage resulting from injury or from disease, for example, pulmonary arterial hypertension (PAH) when the sphingolipid-metabolizing protein is delivered via expression from an Anc80 vector.

Claims

1. A method to minimize damage to a cell/group of cells/tissue in a subject as the result of disease or injury comprising administering to the subject a therapeutically effective amount of an Anc80 viral vector that codes for the expression of a sphingolipid-metabolizing protein.

2. The method of claim 1, wherein the disease is selected from the group consisting of pulmonary arterial hypertension (PAH), stroke, ischemia and reperfusion injury.

3. The method of claim 1, wherein the damage is to pulmonary tissue as the result of pulmonary arterial hypertension (PAH).

4. The method of claim 1, wherein the damage is to cardiac tissue as the result of pulmonary arterial hypertension (PAH).

5. The method of claim 1, wherein said sphingolipid-metabolizing protein is selected from (1) a ceramidase; (2) sphingosine kinase (SPHK); (3) sphingosine-1-phosphate receptor (SIPR); (4) ceramidase kinase (CERK); or a combination of any of (1), (2), (3) and (4).

6. The method of claim 1, wherein said sphingolipid-metabolizing protein is an acid ceramidase.

7. The method of claim 1, wherein said sphingolipid-metabolizing protein is a neutral ceramidase.

8. The method of claim 1, wherein said sphingolipid-metabolizing protein is an alkaline ceramidase.

9. The method of claim 1, wherein the sphingolipid-metabolizing protein is a ceramidase encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

10. The method of claim 1, wherein Anc80 has the nucleotide sequence of SEQ ID NO: 20.

11. A pharmaceutical composition comprising an Anc80 viral vector that codes for expression of a sphingolipid-metabolizing protein and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, wherein said sphingolipid-metabolizing protein is selected from the group consisting of (1) a ceramidase; (2) sphingosine kinase (SPHK); (3) sphingosine-1-phosphate receptor (SIPR); and any combination of 1, 2 and 3.

13. The pharmaceutical composition of claim 11, wherein said sphingolipid-metabolizing protein is an acid ceramidase.

14. The pharmaceutical composition of claim 11, wherein said sphingolipid-metabolizing protein is a neutral ceramidase.

15. The pharmaceutical composition of claim 11, wherein said sphingolipid-metabolizing protein is an alkaline ceramidase.

16. An Anc80 viral vector that codes for expression of a sphingolipid-metabolizing protein for use in the mitigation of tissue damage resulting from disease or injury.

17. The Anc80 viral vector of claim 16, wherein the disease is selected from the group consisting of pulmonary arterial hypertension (PAH), stroke, ischemia and reperfusion injury.

18. The Anc80 viral vector of claim 16 for use in the mitigation of tissue damage from pulmonary arterial hypertension (PAH).

19. The Anc80 viral vector of claim 16, wherein the tissue is lung tissue.

20. The Anc80 viral vector of claim 16, wherein the tissue is cardiac tissue.

21. The Anc80 viral vector of claim 16, wherein said sphingolipid-metabolizing protein is acid ceramidase.

22.-28. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a graph showing percent survival for individuals with pulmonary arterial hypertension (PAH)

[0020] FIG. 2 is a graph showing that right ventricle (RV) function predicts mortality in PAH.

[0021] FIG. 3 shows seven year survival estimates of patients in the REVEAL registry. The REVEAL Registry is a multicenter, observational, U.S.-based study of the clinical course and disease management of PAH.

[0022] FIG. 4 shows biodistribution of Anc80 in a rat model. Rats were injected with Anc80 encoding luciferase. 72 hours post injection luciferase activity was assessed using IVIS machine.

[0023] FIG. 5 shows severe pulmonary artery hypertension (PAH) in the left pneumonectomy combined with Sugen rat model. In this model right ventricular systolic pressure and mean pulmonary artery pressure significantly increased after 6 weeks.

[0024] FIG. 6A-6C shows photomicrographs of hematoxylin and eosin (H&E) staining of lung tissue in PAH. Representative photomicrographs of H&E staining of lung tissue. A Normal lung. B-C Pathological vascular remodeling in PAH rats (pneumonectomy and Sugen). The lung shows concentric medial and intimal thickening (white and black arrows) and severe constricted pulmonary vessels.

[0025] FIG. 7 shows hemodynamic data after Anc80 AC intra-tracheal injection. Rats were subjected to PAH induction protocol. On day 0, rats were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and left lung removal. On day 7, pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and developed neointima and smooth muscle hypertrophy. At week 4, PAH induced rats were treated with Anc80 AC (1×10.sup.11 genome copies). At week 6 and 8, animals were validated by MRI for heart function and RV and PA catheterization for pressure measurement. Treated animals with AC Anc80 at 8 weeks showed excellent cardiac function (validated by MRI) and normal PA pressures despite PAH disease present. After AC administration cardiac output increased 32%.

[0026] FIG. 8 shows hemodynamic data after Anc80 AC intra-tracheal injection. Rats were subjected to PAH induction protocol. On day 0 rats were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and Left lung removal. On day 7, pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and developed neointima and smooth muscle hypertrophy. At week 4, PAH induced rats were treated with Anc80 AC (1×10.sup.11 genome copies). At week 6 and 8, animals were validated by MRI for heart function and RV and PA catheterization for pressure measurement. After AC administration right ventricular systolic volume decreased 39%.

[0027] FIG. 9 shows hemodynamic data after Acn80 AC intra-tracheal injection. Rats were subjected to PAH induction protocol. On week 0 Rat were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and Left lung removal. On day 7 pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and develop neointima and smooth muscle hypertrophy. On week 4 PAH induced rats were treated with Anc80 AC (1×10.sup.11genome copies). On week 6 and 8 animal were validated by MRI for heart function and RV and PA catheterization for pressure measurement. After AC administration right ventricular ejection fraction increased in 65%.

[0028] FIG. 10 shows hemodynamic data after Anc80 AC intra-tracheal injection. Rat were subjected to PAH induced protocol. On week 0 Rat were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and Left lung removal. On day 7 pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and develop neointima and smooth muscle hypertrophy. On week 4 PAH induced rats were treated with Anc80 AC (1×10.sup.11 genome copies). On week 6 and 8 animal were validated by MRI for heart function and RV and PA catheterization for pressure measurement. After AC administration mean pulmonary artery pressure decreased 94%.

[0029] FIG. 11 shows hemodynamic data after Anc80 AC intra-tracheal injection. Rat were subjected to PAH induce protocol. On week 0 Rat were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and Left lung removal. On day 7 pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and develop neointima and smooth muscle hypertrophy. On week 4 PAH induced rats were treated with Anc80 AC (1×10.sup.11 genome copies). On week 6 and 8 animal were validated by MRI for heart function and RV and PA catheterization for pressure measurement. After AC administration mean pulmonary vascular resistance decreased 4.8 times.

[0030] FIG. 12 shows MRI images showing heart function after Anc80 AC intra-tracheal injection. Rats were subjected to PAH induction protocol. On week 0, rats were subjected to baseline MRI, RV and PA catheterization to measure the pressure, and left lung removal. On day 7, pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and developed neointima and smooth muscle hypertrophy. On week 4, PAH-induced rats were treated with Anc80 AC (1×10.sup.11 genome copies). On week 6 and 8, animals were evaluated by MRI for heart function and RV and PA catheterization for pressure measurement. Animals treated with Anc80 AC at 8 weeks showed excellent cardiac function.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] All patents, published applications and other references cited herein are hereby incorporated by reference into the present application.

[0032] 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.

[0033] 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.

[0034] The term “ischemic” as it is known in the art refers to a deficiency in the supply of blood to a part of the body (such as the heart, brain or other organ/tissue) that is due to obstruction of the inflow of arterial blood as by the narrowing of arteries by spasm or disease.

[0035] The term “inhibit” or “inhibition” when used in conjunction with a discussion of senescence includes the ability of the sphingolipid-metabolizing proteins of the disclosure to reverse senescence, thereby returning to normal or near normal function.

[0036] 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.

Pulmonary Arterial Hypertension

[0037] Pulmonary arterial hypertension (PAH) is one form of a broader condition known as pulmonary hypertension, which means high blood pressure in the lungs. In PAH, the rise in blood pressure is caused by changes in the cells that line the pulmonary arteries. These changes can cause the walls of the arteries to become stiff and thick, and extra tissue may form. The blood vessels may also become inflamed and tight. In many cases of pulmonary arterial hypertension, the cause is idiopathic (i.e., unknown). Other causes include heart abnormalities present at birth, HIV infection (Group I PAH); left-sided valvular heart disease such as mitral valve or aortic valve disease (Group 2 PAH); chronic obstructive pulmonary disease and other lung disease (Group 3 PAH); connective tissue/autoimmune disorders (such as scleroderma) and others.

[0038] PAH occurs when the very small arteries throughout the lungs narrow in diameter, which increases the resistance to blood flow through the lungs. Over time, the increased blood pressure can damage the heart. A number of diseases and conditions can cause PAH, and symptoms are similar to the symptoms often seen in more common diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and heart failure.

Mitral Valve Prolapse

[0039] Mitral Valve Prolapse (MVP) is a common disorder afflicting at least 2% to 3% of the general population that affects ≈7.8 million individuals in the United States and >176 million people worldwide [Freed L A 1999, Devereux R B, 2001].

[0040] A canine model of a related disease, Myxomatous Mitral Valve Degeneration, MMVD, is used to further understanding of the role of Anc80 delivery of sphingolipid—metabolizing proteins in MVP.

[0041] The present technology is based on the use of sphingolipid metabolizing proteins in order to manipulate the fate of cells post stress-related events and during disease and 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 an 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.

[0042] 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.

[0043] There are also several studies that support association of the signaling lipid, ceramide, and its metabolizing enzymes with cellular and organismal aging and senescence. It has been reported that the intracellular level of ceramide increased during stress related signaling such as cell culture and aging.

[0044] 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 acts as 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.

[0045] 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).

[0046] In this disclosure, we describe a strategy to reduce pulmonary arterial hypertension by 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).

Choice of Vehicle and Duration of Expression Needed

[0047] Methods and compositions for in vivo delivery of a construct that expresses a sphingolipid-metabolizing protein such as ceramidase were explored. For applications where more sustained expression of a sphingolipid metabolizing enzyme is required, expression from an Anc80 vector may be desirable.

[0048] 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.

[0049] 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.

[0050] The present disclosure, therefore, also provides a method for inhibiting or reducing pulmonary arterial hypertension 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.

Sphinqolipid-Metabolizing Proteins

[0051] 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.

[0052] 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. S1 PR, 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.

[0053] Presently, 7 human ceramidases encoded by 7 distinct genes have been cloned: [0054] acid ceramidase (ASAH1)—associated with cell survival; [0055] neutral ceramidase (ASAH2, ASAH2B, ASAH2C)—protective against inflammatory cytokines; [0056] alkaline ceramidase 1 (ACER1)—mediating cell differentiation by controlling the generation of SPH and S1P; [0057] alkaline ceramidase 2 (ACER2)—important for cell proliferation and survival; and [0058] alkaline ceramidase 3 (ACER3).

[0059] The nucleotide sequences for nucleic acids encoding these ceramidases are shown in Table 1.

[0060] 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.

Advantages

[0061] An Anc80 vector encoding acid ceramidase (Anc80.AC) has multiple advantages over other potential anti-apoptotic factors.

Low toxicity

[0062] Low or no toxicity: The AC protein, by itself, is not toxic. Physiological enzymes are not expected to have toxic effects. The biological function of AC is the control of ceramide metabolism has no direct influence other cellular signaling. Treated cells present only a modest increase in AC generation in cells post gene therapy treatment. The AC protein level expressed after treatment is far below extraordinarily high levels reported in aberrant diseased cells with poorly understood mechanisms. The AC protein exists in two forms, and undergoes a transformation from an inactive to active form in the cell. The inactive AC precursor undergoes an auto-self cleavage to the active enzyme, which is responsible for hydrolyzing ceramide to sphingosine. This exquisitely evolved self-regulating mechanism, call the Sphingolipid Rheostat, regulates, by hydrolysis toxic levels of ceramides in the cell after exposure to stress. The transfection of cells with Anc80.AC can increase the cellular reservoir of inactive precursor, thereby allowing physiological sphingolipid levels to regulate the conversion to the active AC enzyme necessary for cellular robustness and organism survival. In addition, Eliyahu lab created mouse model that is constantly overexpressing the AC enzyme (COEAC) in all tissues. The COEAC mice viability provides evidence that AC is a non-toxic protein.

Ease of delivery

[0063] As mentioned, Anc80, an engineered gene therapy vector, is synthetic in nature and 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 be recognized immunologically by antibodies against naturally occurring AAVs. Recently, it has been shown successful, robust, transfection of Anc80 virus into liver, eye and ear tissue in vivo (see Magali Trayssac, Yusuf A. Hannun, and Lina M. Obeid. Role of sphingolipids in senescence: implication in aging and age-related diseases. J. Clin. Inves. 2018;128(7):2702-2712., which is incorporated herein by reference.)

[0064] In one embodiment, Anc80.AC is administered to at-risk tissue by aerosolization of a composition comprising an Anc80 viral vector that codes for the expression of acid ceram idase.

Unique Physiological Function of Acid Ceramidase

[0065] 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 therefore, the only enzyme that can directly decrease the levels of ceramide in cells.

[0066] Table 1 contains the nucleotide sequences to be encoded by the vectors disclosed for use in practicing the method.

TABLE-US-00001 TABLE 1 Gene Open Reading Frame ASAH1 ATGCCGGGCCGGAGTTGCGTCGCCTTAGTC transcript CTCCTGGCTGCCGCCGTCAGCTGTGCCGTC variant 1 GCGCAGCACGCGCCGCCGTGGACAGAGGAC (ACv1) TGCAGAAAATCAACCTATCCTCCTTCAGGA CCAACGTACAGAGGTGCAGTTCCATGGTAC ACCATAAATCTTGACTTACCACCCTACAAA AGATGGCATGAATTGATGCTTGACAAGGCA CCAGTGCTAAAGGTTATAGTGAATTCTCTG AAGAATATGATAAATACATTCGTGCCAAGT GGAAAAATTATGCAGGTGGTGGATGAAAAA TTGCCTGGCCTACTTGGCAACTTTCCTGGC CCTTTGAAGAGGAAATGAAGGGTATTGCCG CTGTTACTGATATACCTTTAGGAGAGATTA TTTCATTCAATATTTTTTATGAATTATTTA CCATTTGTACTTCAATAGTAGCAGAAGACA AAAAAGGTCATCTAATACATGGGAGAAACA TGGATTTTGGAGTATTTCTTGGGTGGAACA TAAATAATGATACCTGGGTCATAACTGAGC AACTAAAACCTTTAACAGTGAATTTGGATT TCCAAAGAAACAACAAAACTGTCTTCAAGG CTTCAAGCTTTGCTGGCTATGTGGGCATGT TAACAGGATTCAAACCAGGACTGTTCAGTC TTACACTGAATGAACGTTTCAGTATAAATG GTGGTTATCTGGGTATTCTAGAATGGATTC TGGGAAAGAAAGATGTCATGTGGATAGGGT TCCTCACTAGAACAGTTCTGGAAAATAGCA CAAGTTATGAAGAAGCCAAGAATTTATTGA CCAAGACCAAGATATTGGCCCCAGCCTACT TTATCCTGGGAGGCAACCAGTCTGGGGAAG GTTGTGTGATTACACGAGACAGAAAGGAAT CATTGGATGTATATGAACTCGATGCTAAGC AGGGTAGATGGTATGTGGTACAAACAAATT ATGACCGTTGGAAACATCCCTTCTTCCTTG ATGATCGCAGAACGCCTGCAAAGATGTGTC TGAACCGCACCAGCCAAGAGAATATCTCAT TTGAAACCATGTATGATGTCCTGTCAACAA AACCTGTCCTCAACAAGCTGACCGTATACA CAACCTTGATAGATGTTACCAAAGGTCAAT TCGAAACTTACCTGCGGGACTGCCCTGACC CTTGTATAGGTTGGTGA (SEQ ID NO: 1) Sphk1 ATGGATCCAGTGGTCGGTTGCGGACGTGGC CTCTTTGGTTTTGTTTTCTCAGCGGGCGGC CCCCGGGGCGTGCTCCCGCGGCCCTGCCGC GTGCTGGTGCTGCTGAACCCGCGCGGCGGC AAGGGCAAGGCCTTGCAGCTCTTCCGGAGT CACGTGCAGCCCCTTTTGGCTGAGGCTGAA ATCTCCTTCACGCTGATGCTCACTGAGCGG CGGAACCACGCGCGGGAGCTGGTGCGGTCG GAGGAGCTGGGCCGCTGGGACGCTCTGGTG GTCATGTCTGGAGACGGGCTGATGCACGAG GTGGTGAACGGGCTCATGGAGCGGCCTGAC TGGGAGACCGCCATCCAGAAGCCCCTGTGT AGCCTCCCAGCAGGCTCTGGCAACGCGCTG GCAGCTTCCTTGAACCATTATGCTGGCTAT GAGCAGGTCACCAATGAAGACCTCCTGACC AACTGCACGCTATTGCTGTGCCGCCGGCTG CTGTCACCCATGAACCTGCTGTCTCTGCAC ACGGCTTCGGGGCTGCGCCTCTTCTCTGTG CTCAGCCTGGCCTGGGGCTTCATTGCTGAT GTGGACCTAGAGAGTGAGAAGTATCGGCGT CTGGGGGAGATGCGCTTCACTCTGGGCACC TTCCTGCGTCTGGCAGCCCTGCGCACCTAC CGCGGCCGACTGGCCTACCTCCCTGTAGGA AGAGTGGGTTCCAAGACACCTGCCTCCCCC GTTGTGGTCCAGCAGGGCCCGGTAGATGCA CACCTTGTGCCACTGGAGGAGCCAGTGCCC TCTCACTGGACAGTGGTGCCCGACGAGGAC TTTGTGCTAGTCCTGGCACTGCTGCACTCG CACCTGGGCAGTGAGATGTTTGCTGCACCC ATGGGCCGCTGTGCAGCTGGCGTCATGCAT CTGTTCTACGTGCGGGCGGGAGTGTCTCGT GCCATGCTGCTGCGCCTCTTCCTGGCCATG GAGAAGGGCAGGCATATGGAGTATGAATGC CCCTACTTGGTATATGTGCCCGTGGTCGCC TTCCGCTTGGAGCCCAAGGATGGGAAAGGT GTGTTTGCAGTGGATGGGGAATTGATGGTT AGCGAGGCCGTGCAGGGCCAGGTGCACCCA AACTACTTCTGGATGGTCAGCGGTTGCGTG GAGCCCCCGCCCAGCTGGAAGCCCCAGCAG ATGCCACCGCCAGAAGAGCCCTTATGA (SEQ ID NO: 2) S1PR2 ATGGGCAGCTTGTACTCGGAGTACCTGAAC CCCAACAAGGTCCAGGAACACTATAATTAT ACCAAGGAGACGCTGGAAACGCAGGAGACG ACCTCCCGCCAGGTGGCCTCGGCCTTCATC GTCATCCTCTGTTGCGCCATTGTGGTGGAA AACCTTCTGGTGCTCATTGCGGTGGCCCGA AACAGCAAGTTCCACTCGGCAATGTACCTG TTTCTGGGCAACCTGGCCGCCTCCGATCTA CTGGCAGGCGTGGCCTTCGTAGCCAATACC TTGCTCTGGCTCTGTCACGCTGAGGCTGAC GCCTGTGCAGTGGTTTGCCCGGGAGGGCTC TGCCTTCATCACGCTCTCGGCCTCTGTCTT CAGCCTCCTGGCCATCGCCATTGAGCGCCA CGTGGCCATTGCCAAGGTCAAGCTGTATGG CAGCGACAAGAGCTGCCGCATGCTTCTGCT CATCGGGGCCTCGTGGCTCATCTCGCTGGT CCTCGGTGGCCTGCCCATCCTTGGCTGGAA CTGCCTGGGCCACCTCGAGGCCTGCTCCAC TGTCCTGCCTCTCTACGCCAAGCATTATGT GCTGTGCGTGGTGACCATCTTCTCCATCAT CCTGTTGGCCATCGTGGCCCTGTACGTGCG CATCTACTGCGTGGTCCGCTCAAGCCACGC TGACATGGCCGCCCCGCAGACGCTAGCCCT GCTCAAGACGGTCACCATCGTGCTAGGCGT CTTTATCGTCTGCTGGCTGCCCGCCTTCAG CATCCTCCTTCTGGACTATGCCTGTCCCGT CCACTCCTGCCCGATCCTCTACAAAGCCCA CTACTTTTTCGCCGTCTCCACCCTGAATTC CCTGCTCAACCCCGTCATCTACACGTGGCG CAGCCGGGACCTGCGGCGGGAGGTGCTTCG GCCGCTGCAGTGCTGGAGGCCGGGGGTGGG GGTGCAAGGACGGAGGCGGGGCGGGACCCC GGGCCACCACCTCCTGCCACTCCGCAGCTC CAGCTCCCTGGAGAGGGGCATGCACATGCC CACGTCACCCACGTTTCTGGAGGGCAACAC GGTGGTCATG (SEQ ID NO: 3) Firefly ATGGCCGATGCTAAGAACATTAAGAAGGGC luciferase CCTGCTCCCTTCTACCCTCTGGAGGATGGC ACCGCTGGCGAGCAGCTGCACAAGGCCATG AAGAGGTATGCCCTGGTGCCTGGCACCATT GCCTTCACCGATGCCCACATTGAGGTGGAC ATCACCTATGCCGAGTACTTCGAGATGTCT GTGCGCCTGGCCGAGGCCATGAAGAGGTAC GGCCTGAACACCAACCACCGCATCGTGGTG TGCTCTGAGAACTCTCTGCAGTTCTTCATG CCAGTGCTGGGCGCCCTGTTCATCGGAGTG GCCGTGGCCCCTGCTAACGACATTTACAAC GAGCGCGAGCTGCTGAACAGCATGGGCATT TCTCAGCCTACCGTGGTGTTCGTGTCTAAG AAGGGCCTGCAGAAGATCCTGAACGTGCAG AAGAAGCTGCCTATCATCCAGAAGATCATC ATCATGGACTCTAAGACCGACTACCAGGGC TTCCAGAGCATGTACACATTCGTGACATCT CATCTGCCTCCTGGCTTCAACGAGTACGAC TTCGTGCCAGAGTCTTTCGACAGGGACAAA ACCATTGCCCTGATCATGAACAGCTCTGGG TCTACCGGCCTGCCTAAGGGCGTGGCCCTG CCTCATCGCACCGCCTGTGTGCGCTTCTCT CACGCCCGCGACCCTATTTTCGGCAACCAG ATCATCCCCGACACCGCTATTCTGAGCGTG GTGCCATTCCACCACGGCTTCGGCATGTTC ACCACCCTGGGCTACCTGATTTGCGGCTTT CGGGTGGTGCTGATGTACCGCTTCGAGGAG GAGCTGTTCCTGCGCAGCCTGCAAGACTAC AAAATTCAGTCTGCCCTGCTGGTGCCAACC CTGTTCAGCTTCTTCGCTAAGAGCACCCTG ATCGACAAGTACGACCTGTCTAACCTGCAC GAGATTGCCTCTGGCGGCGCCCCACTGTCT AAGGAGGTGGGCGAAGCCGTGGCCAAGCGC TTTCATCTGCCAGGCATCCGCCAGGGCTAC GGCCTGACCGAGACAACCAGCGCCATTCTG ATTACCCCAGAGGGCGACGACAAGCCTGGC GCCGTGGGCAAGGTGGTGCCATT CTTCGAGGCCAAGGTGGTGGACCTGGACAC CGGCAAGACCCTGGGAGTGAACCAGCGCGG CGAGCTGTGTGTGCGCGGCCCTATGATTAT GTCCGGCTACGTGAATAACCCTGAGGCCAC AAACGCCCTGATCGACAAGGACGGCTGGCT GCACTCTGGCGACATTGCCTACTGGGACGA GGACGAGCACTTCTTCATCGTGGACCGCCT GAAGTCTCTGATCAAGTACAAGGGCTACCA GGTGGCCCCAGCCGAGCTGGAGTCTATCCT GCTGCAGCACCCTAACATTTTCGACGCCGG AGTGGCCGGCCTGCCCGACGACGATGCCGG CGAGCTGCCTGCCGCCGTCGTCGTGCTGGA ACACGGCAAGACCATGACCGAGAAGGAGAT CGTGGACTATGTGGCCAGCCAGGTGACAAC CGCCAAGAAGCTGCGCGGCGGAGTGGTGTT CGTGGACGAGGTGCCCAAGGGCCTGACCGG CAAGCTGGACGCCCGCAAGATCCGCGAGAT CCTGATCAAGGCTAAGAAAGGCGGCAAGAT CGCCGTGTAA (SEQ ID NO: 4) nGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACC GGGGTGGTGCCCATCCTGGTCGAGCTGGAC GGCGACGTAAACGGCCACAAGTTCAGCGTG TCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCGTGGCCCAC CCTCGTGACCACCCTGACCTACGGCGTGCA GTGCTTCAGCCGCTACCCCGACCACATGAA GCAGCACGACTTCTTCAAGTCCGCCATGCC CGAAGGCTACGTCCAGGAGCGCACCATCTT CTTCAAGGACGACGGCAACTACAAGACCCG CGCCGAGGTGAAGTTCGAGGGCGACACCCT GGTGAACCGCATCGAGCTGAAGGGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAA CGTCTATATCATGGCCGACAAGCAGAAGAA CGGCATCAAGGTGAACTTCAAGATCCGCCA CAACATCGAGGACGGCAGCGTGCAGCTCGC CGACCACTACCAGCAGAACACCCCCATCGG CGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCACCCAGTCCGCCCTGAGCAA AGACCCCAACGAGAAGCGCGATCACATGGT CCTGCTGGAGTTCGTGACCGCCGCCGGGAT CACTCTCGGCATGGACGAGCTGTACAAGGG AGATCCAAAAAAGAAGAGAAAGGTAGGCGA TCCAAAAAAGAAGAGAAAGGTAGGTGATCC AAAAAAGAAGAGAAAGGTATAA (SEQ ID NO: 5) ASAH2 ATGAACTGCTGCATCGGGCTGGGAGAGAAA transcript GCTCGCGGGTCCCACCGGGCCTCCTACCCA variant 2 AGTCTCAGCGCGCTTTTCACCGAGGCCTCA (ACv2) ATTCTGGGATTTGGCAGCTTTGCTGTGAAA GCCCAATGGACAGAGGACTGCAGAAAATCA ACCTATCCTCCTTCAGGACCAACGTACAGA GGTGCAGTTCCATGGTACACCATAAATCTT GACTTACCACCCTACAAAAGATGGCATGAA TTGATGCTTGACAAGGCACCAGTGCTAAAG GTTATAGTGAATTCTCTGAAGAATATGATA AATACATTCGTGCCAAGTGGAAAAATTATG CAGGTGGTGGATGAAAAATTGCCTGGCCTA CTTGGCAACTTTCCTGGCCCTTTTGAAGAG GAAATGAAGGGTATTGCCGCTGTTACTGAT ATACCTTTAGGAGAGATTATTTCATTCAAT ATTTTTTATGAATTATTTACCATTTGTACT TCAATAGTAGCAGAAGACAAAAAAGGTCAT CTAATACATGGGAGAAACATGGATTTTGGA GTATTTCTTGGGTGGAACATAAATAATGAT ACCTGGGTCATAACTGAGCAACTAAAACCT TTAACAGTGAATTTGGATTTCCAAAGAAAC AACAAAACTGTCTTCAAGGCTTCAAGCTTT GCTGGCTATGTGGGCATGTTAACAGGATTC AAACCAGGACTGTTCAGTCTTACACTGAAT GAACGTTTCAGTATAAATGGTGGTTATCTG GGTATTCTAGAATGGATTCTGGGAAAGAAA GATGTCATGTGGATAGGGTTCCTCACTAGA ACAGTTCTGGAAAATAGCACAAGTTATGAA GAAGCCAAGAATTTATTGACCAAGACCAAG ATATTGGCCCCAGCCTACTTTATCCTGGGA GGCAACCAGTCTGGGGAAGGTTGTGTGATT ACACGAGACAGAAAGGAATCATTGGATGTA TATGAACTCGATGCTAAGCAGGGTAGATGG TATGTGGTACAAACAAATTATGACCGTTGG AAACATCCCTTCTTCCTTGATGATCGCAGA ACGCCTGCAAAGATGTGTCTGAACCGCACC AGCCAAGAGAATATCTCATTTGAAACCATG TATGATGTCCTGTCAACAAAACCTGTCCTC AACAAGCTGACCGTATACACAACCTTGATA GATGTTACCAAAGGTCAATTCGAAACTTAC CTGCGGGACTGCCCTGACCCTTGTATAGGT TGGTGA (SEQ ID NO: 6) ASAH1 ATGAACTGCTGCATCGGGCTGGGAGAGAAA transcript GCTCGCGGGTCCCACCGGGCCTCCTACCCA variant 3 AGTCTCAGCGCGCTTTTCACCGAGGCCTCA ATTCTGGGATTTGGCAGCTTTGCTGTGAAA GCCCAATGGACAGAGGACTGCAGAAAATCA ACCTATCCTCCTTCAGGACCAACTGTCTTC CCTGCTGTTATAAGGTACAGAGGTGCAGTT CCATGGTACACCATAAATCTTGACTTACCA CCCTACAAAAGATGGCATGAATTGATGCTT GACAAGGCACCAGTGCCTGGCCTACTTGGC AACTTTCCTGGCCCTTTTGAAGAGGAAATG AAGGGTATTGCCGCTGTTACTGATATACCT TTAGGAGAGATTATTTCATTCAATATTTTT TATGAATTATTTACCATTTGTACTTCAATA GTAGCAGAAGACAAAAAAGGTCATCTAATA CATGGGAGAAACATGGATTTTGGAGTATTT TCTTGGGTGGAACATAAATAATGATACCTG GGTCATAACTGAGCAACTAAAACCTTTAAC AGTGAATTTGGATTTCCAAAGAAACAACAA AACTGTCTTCAAGGCTTCAAGCTTTGCTGG CTATGTGGGCATGTTAACAGGATTCAAACC AGGACTGTTCAGTCTTACACTGAATGAACG TTTCAGTATAAATGGTGGTTATCTGGGTAT TCTAGAATGGATTCTGGGAAAGAAAGATGT CATGTGGATAGGGTTCCTCACTAGAACAGT TCTGGAAAATAGCACAAGTTATGAAGAAGC CAAGAATTTATTGACCAAGACCAAGATATT GGCCCCAGCCTACTTTATCCTGGGAGGCAA CCAGTCTGGGGAAGGTTGTGTGATTACACG AGACAGAAAGGAATCATTGGATGTATATGA ACTCGATGCTAAGCAGGGTAGATGGTATGT GGTACAAACAAATTATGACCGTTGGAAACA TCCCTTCTTCCTTGATGATCGCAGAACGCC TGCAAAGATGTGTCTGAACCGCACCAGCCA AGAGAATATCTCATTTGAAACCATGTATGA TGTCCTGTCAACAAAACCTGTCCTCAACAA GCTGACCGTATACACAACCTTGATAGATGT TACCAAAGGTCAATTCGAAACTTACCTGCG GGACTGCCCTGACCCTTGTATAGGTTGGTG A (SEQ ID NO: 7) ASAH2 ATGGCCAAACGCACCTTCTCTAACTTGGAG transcript ACATTCCTGATTTTCCTCCTTGTAATGATG variant 1 AGTGCCATCACAGTGGCCCTTCTCAGCCTC TTGTTTATCACCAGTGGGACCATTGAAAAC CACAAAGATTTAGGAGGCCATTTTTTTTCA ACCACCCAAAGCCCTCCAGCCACCCAGGGC TCCACAGCTGCCCAACGCTCCACAGCCACC CAGCATTCCACAGCCACCCAGAGCTCCACA GCCACTCAAACTTCTCCAGTGCCTTTAACC CCAGAGTCTCCTCTATTTCAGAACTTCAGT GGCTACCATATTGGTGTTGGACGAGCTGAC TGCACAGGACAAGTAGCAGATATCAATTTG ATGGGCTATGGCAAATCCGGCCAGAATGCA CAGGGCATCCTCACCAGGCTATACAGTCGT GCCTTCATCATGGCAGAACCTGATGGGTCC AATCGAACAGTGTTTGTCAGCATCGACATA GGCATGGTATCACAAAGGCTCAGGCTGGAG GTCCTGAACAGACTGCAGAGTAAATATGGC TCCCTGTACAGAAGAGATAATGTCATCCTG AGTGGCACTCACACTCATTCAGGTCCTGCA GGATATTTCCAGTATACCGTGTTTGTAATT GCCAGTGAAGGATTTAGCAATCAAACTTTT CAGCACATGGTCACTGGTATCTTGAAGAGC ATTGACATAGCACACACAAATATGAAACCA GGCAAAATCTTCATCAATAAAGGAAATGTG GATGGTGTGCAGATCAACAGAAGTCCGTAT TCTTACCTTCAAAATCCGCAGTCAGAGAGA GCAAGGTATTCTTCAAATACAGACAAGGAA ATGATAGTTTTGAAAATGGTAGATTTGAAT GGAGATGACTTGGGCCTTATCAGCTGGTTT GCCATCCACCCGGTCAGCATGAACAACAGT AACCATCTTGTAAACAGTGACAATGTGGGC TATGCATCTTACCTGCTTGAGCAAGAGAAG AACAAAGGATATCTACCTGGACAGGGGCCA TTTGTAGCAGCCTTTGCTTCATCAAACCTA GGAGATGTGTCCCCCAACATTCTTGGACCA CGTTGCATCAACACAGGAGAGTCCTGTGAT AACGCCAATAGCACTTGTCCCATTGGTGGG CCTAGCATGTGCATTGCTAAGGGACCTGGA CAGGATATGTTTGACAGCACACAAATTATA GGACGGGCCATGTATCAGAGAGCAAAGGAA CTCTATGCCTCTGCCTCCCAGGAGGTAACA GGACCACTGGCTTCAGCACACCAGTGGGTG GATATGACAGATGTGACTGTCTGGCTCAAT TCCACACATGCATCAAAAACATGTAAACCA GCATTGGGCTACAGTTTTGCAGCTGGCACT ATTGATGGAGTTGGAGGCCTCAATTTTACA CAGGGGAAAACAGAAGGGGATCCAMTTGGG ACACCATTCGGGACCAGATCCTGGGAAAGC CATCTGAAGAAATTAAAGAATGTCATAAAC CAAAGCCCATCCTTCTTCACACCGGAGAAC TATCAAAACCTCACCCCTGGCATCCAGACA TTGTTGATGTTCAGATTATTACCCTTGGGT CCTTGGCCATAACTGCCATCCCCGGGGAGT TTACGACCATGTCTGGACGAAGACTTCGAG AGGCAGTTCAAGCAGAATTTGCATCTCATG GGATGCAGAACATGACTGTTGTTATTTCAG GTCTATGCAACGTCTATACACATTACATTA CCACTTATGAAGAATACCAGGCTCAGCGAT ATGAGGCAGCATCGACAATTTATGGACCGC ACACATTATCTGCTTACATTCAGCTCTTCA GAAACCTTGCTAAGGCTATTGCTACGGACA CGGTAGCCAACCTGAGCAGAGGTCCAGAAC CTCCCTTTTTCAAACAATTAATAGTTCCAT TAATTCCTAGTATTGTGGATAGAGCACCAA AAGGCAGAACTTTCGGGGATGTCCTGCAGC CAGCAAAACCTGAATACAGAGTGGGGGAAG TTGCTGAAGTTATATTTGTAGGTGCTAACC CGAAGAATTCAGTACAAAACCAGACCCATC AGACCTTCCTCACTGTGGAGAAATATGAGG CTACTTCAACATCGTGGCAGATAGTGTGTA ATGATGCCTCCTGGGAGACTCGTTTTTATT GGCACAAGGGACTCCTGGGTCTGAGTAATG CAACAGTGGAATGGCATATTCCAGACACTG CCCAGCCTGGAATCTACAGAATAAGATATT TTGGACACAATCGGAAGCAGGACATTCTGA AGCCTGCTGTCATACTTTCATTTGAAGGCA CTTCCCCGGCTTTTGAAGTTGTAACTATTT AGTGA (SEQ ID NO: 8) ASAH2 ATGGCCAAACGCACCTTCTCTAACTTGGAG transcript ACATTCCTGATTTTCCTCCTTGTAATGATG variant 2 AGTGCCATCACAGTGGCCCTTCTCAGCCTC TTGTTTATCACCAGTGGGACCATTGAAAAC CACAAAGATTTAGGAGGCCATTTTTTTTCA ACCACCCAAAGCCCTCCAGCCACCCAGGGC TCCACAGCTGCCCAACGCTCCACAGCCACC CAGCATTCCACAGCCACCCAGAGCTCCACA GCCACTCAAACTTCTCCAGTGCCTTTAACC CCAGAGTCTCCTCTATTTCAGAACTTCAGT GGCTACCATATTGGTGTTGGACGAGCTGAC TGCACAGGACAAGTAGCAGATATCAATTTG ATGGGCTATGGCAAATCCGGCCAGAATGCA CAGGGCATCCTCACCAGGCTATACAGTCGT GCCTTCATCATGGCAGAACCTGATGGGTCC AATCGAACAGTGTTTGTCAGCATCGACATA GGCATGGTATCACAAAGGCTCAGGCTGGAG GTCCTGAACAGAC TGCAGAGTAAATATGGCTCCCTGTACAGAA GAGATAATGTCATCCTGAGTGGCACTCACA CTCATTCAGGTCCTGCAGGATATTTCCAGT ATACCGTGTTTGTAATTGCCAGTGAAGGAT TTAGCAATCAAACTTTTCAGCACATGGTCA CTGGTATCTTGAAGAGCATTGACATAGCAC ACACAAATATGAAACCAGGCAAAATCTTCA TCAATAAAGGAAATGTGGATGGTGTGCAGA TCAACAGAAGTCCGTATTCTTACCTTCAAA ATCCGCAGTCAGAGAGAGCAAGGTATTCTT CAAATACAGACAAGGAAATGATAGTTTTGA AAATGGTAGATTTGAATGGAGATGACTTGG GCCTTATCAGCTGGTTTGCCATCCACCCGG TCAGCATGAACAACAGTAACCATCTTGTAA ACAGTGACAATGTGGGCTATGCATCTTACC TGCTTGAGCAAGAGAAGAACAAAGGATATC TACCTGGACAGGGGCCATTTGTAGCAGCCT TTGCTTCATCAAACCTAGGAGATGTGTCCC CCAACATTCTTGGACCACGTTGCATCAACA CAGGAGAGTCCTGTGATAACGCCAATAGCA CTTGTCCCATTGGTGGGCCTAGCATGTGCA TTGCTAAGGGACCTGGACAGGATATGTTTG ACAGCACACAAATTATAGGACGGGCCATGT ATCAGAGAGCAAAGTCAAAAACATGTAAAC CAGCATTGGGCTACAGTTTTGCAGCTGGCA CTATTGATGGAGTTGGAGGCCTCAATTTTA CACAGGGGAAAACAGAAGGGGATCCATTTT GGGACACCATTCGGGACCAGATCCTGGGAA AGCCATCTGAAGAAATTAAAGAATGTCATA AACCAAAGCCCATCCTTCTTCACACCGGAG AACTATCAAAACCTCACCCCTGGCATCCAG ACATTGTTGATGTTCAGATTATTACCCTTG GGTCCTTGGCCATAACTGCCATCCCCGGGG AGTTTACGACCATGTCTGGACGAAGACTTC GAGAGGCAGTTCAAGCAGAATTTGCATCTC ATGGGATGCAGAACATGACTGTTGTTATTT CAGGTCTATGCAACGTCTATACACATTACA TTACCACTTATGAAGAATACCAGGCTCAGC GATATGAGGCAGCATCGACAATTTATGGAC CGCACACATTATCTGCTTACATTCAGCTCT TCAGAAACCTTGCTAAGGCTATTGCTACGG ACACGGTAGCCAACCTGAGCAGAGGTCCAG AACCTCCCTTTTTCAAACAATTAATAGTTC CATTAATTCCTAGTATTGTGGATAGAGCAC CAAAAGGCAGAACTTTCGGGGATGTCCTGC AGCCAGCAAAACCTGAATACAGAGTGGGGG AAGTTGCTGAAGTTATATTTGTAGGTGCTA ACCCGAAGAATTCAGTACAAAACCAGACCC ATCAGACCTTCCTCACTGTGGAGAAATATG AGGCTACTTCAACATCGTGGCAGATAGTGT GTAATGATGCCTCCTGGGAGACTCGTTTTT TATTGGCACAAGGGACTCCTGGGTCTGAGT AATGCAACAGTGGAATGGCATATTCCAGAC ACTGCCCAGCCTGGAATCTACAGAATAAGA TATTTTGGACACAATCGGAAGCAGGACATT CTGAAGCCTGCTGTCATACTTTCATTTGAA GGCACTTCCCCGGCTTTTGAAGTTGTAACT ATTTAGTGA (SEQ ID NO: 9) ASAH2B ATGAGGCAGCATCGACAATTTATGGACCGC transcript ACGCATTATCTGCTTACATTCAGCTCTTCA variant 1 GAAACCTTGCTAAGGCTATTGCTACGTATT GTGGATAGAGCACCAAAAGGCAGAACTTTC GGGGATGTCCTGCAGCCAGCAAAACCTGAA TACAGAGTGGGGGAAGTTGCTGAAGTTATA TTTGTAGGTGCTAACCCGAAGAATTCAGTA CAAAACCAGACCCATCAGACCTTCCTCACT GTGGAGAAATATGAGGCTACTTCAACATCG TGGCAGATAGTGTGTAATGATGCCTCCTGG GAGACTCGTTTTTATTGGCACAAGGGACTC CTGGGTCTGAGTAATGCAACAGTGGAATGG CATATTCCAGACACTGCCCAGCCTGGAATC TACAGAATAAGATATTTTGGACACAATCGG AAGCAGGACATTCTGAAGCCTGCTGTCATA CTTTCATTTGAAGGCACTTCCCCGGCTTTT GAAGTTGTAACTATTTAGTGA (SEQ ID NO: 10) ASAH2B ATGGTAGCCAACCTGAGCAGAGGTCCAGAA transcript CCTCCCTTTTTCAAACAATTAATAGTTCCA variant 3 TTAATTCCTAGTATTGTGGATAGAGCACCA AAAGGCAGAACTTTCGGGGATGTCCTGCAG CCAGCAAAACCTGAATACAGAGTGGGGGAA GTTGCTGAAGTTATATTTGTAGGTGCTAAC CCGAAGAATTCAGTACAAAACCAGACCCAT CAGACCTTCCTCACTGTGGAGAAATATGAG GCTACTTCAACATCGTGGCAGATAGTGTGT AATGATGCCTCCTGGGAGACTCGTTTTTAT TGGCACAAGGGACTCCTGGGTCTGAGTAAT GCAACAGTGGAATGGCATATTCCAGACACT GCCCAGCCTGGAATCTACAGAATAAGATAT TTTGGACACAATCGGAAGCAGGACATTCTG AAGCCTGCTGTCATACTTTCATTTGAAGGC ACTTCCCCGGCTTTTGAAGTTGTAACTATT TAGTGAATGGTAGCCAACCTGAGCAGAGGT CCAGAACCTCCCTTTTTCAAACAATTAATA GTTCCATTAATTCCTAGTATTGTGGATAGA GCACCAAAAGGCAGAACTTTCGGGGATGTC CTGCAGCCAGCAAAACCTGAATACAGAGTG GGGGAAGTTGCTGAAGTTATATTTGTAGGT GCTAACCCGAAGAATTCAGTACAAAACCAG ACCCATCAGACCTTCCTCACTGTGGAGAAA TATGAGGCTACTTCAACATCGTGGCAGATA GTGTGTAATGATGCCTCCTGGGAGACTCGT TTTTATTGGCACAAGGGACTCCTGGGTCTG AGTAATGCAACAGTGGAATGGCATATTCCA GACACTGCCCAGCCTGGAATCTACAGAATA AGATATTTTGGACACAATCGGAAGCAGGAC ATTCTGAAGCCTGCTGTCATACTTTCATTT GAAGGCACTTCCCCGGCTTTTGAAGTTGTA ACTATTTAGTGA (SEQ ID NO: 11) ASAH2B ATGGTAGCCAACCTGAGCAGAGGTCCAGAA transcript CCTCCCTTTTTCAAACAATTAATAGTTCCA variant 4 TTAATTCCTAGTATTGTGGATAGAGCACCA AAAGGCAGAACTTTCGGGGATGTCCTGCAG CCAGCAAAACCTGAATACAGAGTGGGGGAA GTTGCTGAAGTTATATTTGTAGGTGCTAAC CCGAAGAATTCAGTACAAAACCAGACCCAT CAGACCTTCCTCACTGTGGAGAAATATGAG GCTACTTCAACATCGTGGCAGATAGTGTGT AATGATGCCTCCTGGGAGACTCGTTTTTAT TGGCACAAGGGACTCCTGGGTCTGAGTAAT GCAACAGTGGAATGGCATATTCCAGACACT GCCCAGCCTGGAATCTACAGAATAAGATAT TTTGGACACAATCGGAAGCAGGACATTCTG AAGCCTGCTGTCATACTTTCATTTGAAGGC ACTTCCCCGGCTTTTGAAGTTGTAACTATT TAG (SEQ ID NO: 12) ACER1 ATGCCTAGCATCTTCGCCTATCAGAGCTCC GAGGTGGACTGGTGTGAGAGCAACTTCCAG TACTCGGAGCTGGTGGCCGAGTTCTACAAC ACGTTCTCCAATATCCCCTTCTTCATCTTC GGGCCACTGATGATGCTCCTGATGCACCCG TATGCCCAGAAGCGCTCCCGCTACATTTAC GTTGTCTGGGTCCTCTTCATGATCATAGGC CTGTTCTCCATGTATTTCCACATGACGCTC AGCTTCCTGGGCCAGCTGCTGGACGAGATC GCCATCCTGTGGCTCCTGGGCAGTGGCTAT AGCATATGGATGCCCCGCTGCTATTTCCCC TCCTTCCTTGGGGGGAACAGGTCCCAGTTC ATCCGCCTGGTCTTCATCACCACTGTGGTC AGCACCCTTCTGTCCTTCCTGCGGCCCACG GTCAACGCCTACGCCCTCAACAGCATTGCC CTGCACATTCTCTACATCGTGTGCCAGGAG TACAGGAAGACCAGCAATAAGGAGCTTCGG CACCTGATTGAGGTCTCCGTGGTTTTATGG GCTGTTGCTCTGACCAGCTGGATCAGTGAC CGTCTGCTTTGCAGCTTCTGGCAGAGGATT CATTTCTTCTATCTGCACAGCATCTGGCAT GTGCTCATCAGCATCACCTTCCCTTATGGC ATGGTCACCATGGCCTTGGTGGATGCCAAC TATGAGATGCCAGGTGAAACCCTCAAAGTC CGCTACTGGCCTCGGGACAGTTGGCCCGTG GGGCTGCCCTACGTGGAAATCCGGGGTGAT GACAAGGACTGCTGA (SEQ ID NO: 13) ACER2 ATGGGCGCCCCGCACTGGTGGGACCAGCTG CAGGCTGGTAGCTCGGAGGTGGACTGGTGC GAGGACAACTACACCATCGTGCCTGCTATC GCCGAGTTCTACAACACGATCAGCAATGTC TTATTTTTCATMTACCGCCCATCTGCATGT GCTTGTTTCGTCAGTATGCAACATGCTTCA ACAGTGGCATCTACTTAATCTGGACTCTTT TGGTTGTAGTGGGAATTGGATCCGTCTACT TCCATGCAACCCTTAGTTTCTTGGGTCAGA TGCTTGATGAACTTGCAGTCCTTTGGGTTC TGATGTGTGCTTTGGCCATGTGGTTCCCCA GAAGGTATCTACCAAAGATCTTTCGGAATG ACCGGGGTAGGTTCAAGGTGGTGGTCAGTG TCCTGTCTGCGGTTACGACGTGCCTGGCAT TTGTCAAGCCTGCCATCAACAACATCTCTC TGATGACCCTGGGAGTTCCTTGCACTGCAC TGCTCATCGCAGAGCTAAAGAGGTGTGACA ACATGCGTGTGTTTAAGCTGGGCCTCTTCT CGGGCCTCTGGTGGACCCTGGCCCTGTTCT GCTGGATCAGTGACCGAGCTTTCTGCGAGC TGCTGTCATCCTTCAACTTCCCCTACCTGC ACTGCATGTGGCACATCCTCATCTGCCTTG CTGCCTACCTGGGCTGTGTATGCTTTGCCT ACTTTGATGCTGCCTCAGAGATTCCTGAGC AAGGCCCTGTCATCAAGTTCTGGCCCAATG AGAAATGGGCCTTCATTGGTGTCCCCTATG TGTCCCTCCTGTGTGCCAACAAGAAATCAT CAGTCAAGATCACGTGA (SEQ ID NO: 14) ACER3 ATGGCTCCGGCCGCGGACCGAGAGGGCTAC transcript TGGGGCCCCACGACCTCCACGCTGGACTGG variant 1 TGCGAGGAGAACTACTCCGTGACCTGGTAC ATCGCCGAGTTCTGGAATACAGTGAGTAAC CTGATCATGATTATACCTCCAATGTTCGGT GCAGTTCAGAGTGTTAGAGACGGTCTGGAA AAGCGGTACATTGCTTCTTATTTAGCACTC ACAGTGGTAGGAATGGGATCCTGGTGCTTC CACATGACTCTGAAATATGAAATGCAGCTA TTGGATGAACTCCCAATGATATACAGCTGT TGCATATTTGTGTACTGCATGTTTGAATGT TTCAAGATCAAGAACTCAGTAAACTACCAT CTGCTTTTTACCTTAGTTCTATTCAGTTTA ATAGTAACCACAGTTTACCTTAAGGTAAAA GAGCCGATATTCCATCAGGTCATGTATGGA ATGTTGGTCTTTACATTAGTACTTCGATCT ATTTATATTGTTACATGGGTTTATCCATGG CTTAGAGGACTGGGTTATACATCATTGGGT ATATTTMATTGGGATTTTTATTTTGGAATA TAGATAACATATTTTGTGAGTCACTGAGGA ACTTTCGAAAGAAGGTACCACCTATCATAG GTATTACCACACAATTTCATGCATGGTGGC ATATTTTAACTGGCCTTGGTTCCTATCTTC ACATCCTTTTCAGTTTGTATACAAGAACAC TTTACCTGAGATATAGGCCAAAAGTGAAGT TTCTCTTTGGAATCTGGCCAGTGATCCTGT TTGAGCCTCTCAGGAAGCATVGA (SEQ ID NO: 15) ACER3 ATGGCTCCGGCCGCGGACCGAGAGGGCTAC transcript TGGGGCCCCACGACCTCCACGCTGGACTGG variant 2 TGCGAGGAGAACTACTCCGTGACCTGGTAC ATCGCCGAGTTCTTGGTAGGAATGGGATCC TGGTGCTTCCACATGACTCTGAAATATGAA ATGCAGCTATTGGATGAACTCCCAATGATA TACAGCTGTTGCATATTTGTGTACTGCATG TTTGAATGTTTCAAGATCAAGAACTCAGTA AACTACCATCTGCTTTTTACCTTAGTTCTA TTCAGTTTAATAGTAACCACAGTTTACCTT AAGGTAAAAGAGCCGATATTCCATCAGGTC ATGTATGGAATGTTGGTCTTTACATTAGTA CTTCGATCTATTTATATTGTTACATGGGTT TATCCATGGCTTAGAGGACTGGGTTATACA TCATTGGGTATATTTTTATTGGGATTTTTA TTTTGGAATATAGATAACATATTTTGTGAG TCACTGAGGAACTTTCGAAAGAAGGTACCA CCTATCATAGGTATTACCACACAATTTCAT GCATGGTGGCATATTTTAACTGGCCTTGGT TCCTATCTTCACATCCTTTTCAGTTTGTAT ACAAGAACACTTTACCTGAGATATAGGCCA AAAGTGAAGTTTCTCTTTGGAATCTGGCCA GTGATCCTGTTTGAGCCTCTCAGGAAGCAT GA(SEQIDN0:16) ACER3 ATGATATACAGCTGTTGCATATTTGTGTAC transcript TGCATGTTTGAATGTTTCAAGATCAAGAAC variant 3 TCAGTAAACTACCATCTGCTTTTTACCTTA GTTCTATTCAGTTTAATAGTAACCACAGTT TACCTTAAGGTAAAAGAGCCGATATTCCAT CAGGTCATGTATGGAATGTTGGTCTTTACA TTAGTACTTCGATCTATTTATATTGTTACA TGGGTTTATCCATGGCTTAGAGGACTGGGT TATACATCATTGGGTATATTTTTATTGGGA TTTTTATTTTGGAATATAGATAACATATTT TGTGAGTCACTGAGGAACTTTCGAAAGAAG GTACCACCTATCATAGGTATTACCACACAA TTTCATGCATGGTGGCATATTTTAACTGGC CTTGGTTCCTATCTTCACATCCTTTTCAGT TTGTATACAAGAACACTTTACCTGAGATAT AGGCCAAAAGTGAAGTTTCTCTTTGGAATC TGGCCAGTGATCCTGTTTGAGCCTCTCAGG AAGCATTGA (SEQ ID NO: 17) Sphk2 ATGAATGGACACCTTGAAGCAGAGGAGCAG CAGGACCAGAGGCCAGACCAGGAGCTGACC GGGAGCTGGGGCCACGGGCCTAGGAGCACC CTGGTCAGGGCTAAGGCCATGGCCCCGCCC CCACCGCCACTGGCTGCCAGCACCCCGCTC CTCCATGGCGAGTTTGGCTCCTACCCAGCC CGAGGCCCACGCTTTGCCCTCACCCTTACA TCGCAGGCCCTGCACATACAGCGGCTGCGC CCCAAACCTGAAGCCAGGCCCCGGGGTGGC CTGGTCCCGTTGGCCGAGGTCTCAGGCTGC TGCACCCTGCGAAGCCGCAGCCCCTCAGAC TCAGCGGCCTACTTCTGCATCTACACCTAC CCTCGGGGCCGGCGCGGGGCCCGGCGCAGA GCCACTCGCACCTTCCGGGCAGATGGGGCC GCCACCTACGAAGAGAACCGTGCCGAGGCC CAGCGCTGGGCCACTGCCCTCACCTGTCTG CTCCGAGGACTGCCACTGCCCGGGGATGGG GAGATCACCCCTGACCTGCTACCTCGGCCG CCCCGGTTGCTTCTATTGGTCAATCCCTTT GGGGGTCGGGGCCTGGCCTGGCAGTGGTGT AAGAACCACGTGCTTCCCATGATCTCTGAA GCTGGGCTGTCCTTCAACCTCATCCAGACA GAACGACAGAACCACGCCCGGGAGCTGGTC CAGGGGCTGAGCCTGAGTGAGTGGGATGGC ATCGTCACGGTCTCGGGAGACGGGCTGCTC CATGAGGTGCTGAACGGGCTCCTAGATCGC CCTGACTGGGAGGAAGCTGTGAAGATGCCT GTGGGCATCCTCCCCTGCGGCTCGGGCAAC GCGCTGGCCGGAGCAGTGAACCAGCACGGG GGATTTGAGCCAGCCCTGGGCCTCGACCTG TTGCTCAACTGCTCACTGTTGCTGTGCCGG GGTGGTGGCCACCCACTGGACCTGCTCTCC GTGACGCTGGCCTCGGGCTCCCGCTGTTTC TCCTTCCTGTCTGTGGCCTGGGGCTTCGTG TCAGATGTGGATATCCAGAGCGAGCGCTTC AGGGCCTTGGGCAGTGCCCGCTTCACACTG GGCACGGTGCTGGGCCTCGCCACACTGCAC ACCTACCGCGGACGCCTCTCCTACCTCCCC GCCACTGTGGAACCTGCCTCGCCCACCCCT GCCCATAGCCTGCCTCGTGCCAAGTCGGAG CTGACCCTAACCCCAGACCCAGCCCCGCCC ATGGCCCACTCACCCCTGCATCGTTCTGTG TCTGACCTGCCTCTTCCCCTGCCCCAGCCT GCCCTGGCCTCTCCTGGCTCGCCAGAACCC CTGCCCATCCTGTCCCTCAACGGTGGGGGC CCAGAGCTGGCTGGGGACTGGGGTGGGGCT GGGGATGCTCCGCTGTCCCCGGACCCACTG CTGTCTTCACCTCCTGGCTCTCCCAAGGCA GCTCTACACTCACCCGTCTCCGAAGGGGCC CCCGTAATTCCCCCATCCTCTGGGCTCCCA CTTCCCACCCCTGATGCCCGGGTAGGGGCC TCCACCTGCGGCCCGCCCGACCACCTGCTG CCTCCGCTGGGCACCCCGCTGCCCCCAGAC TGGGTGACGCTGGAGGGGGACTTTGTGCTC ATGTTGGCCATCTCGCCCAGCCACCTAGGC GCTGACCTGGTGGCAGCTCCGCATGCGCGC TTCGACGACGGCCTGGTGCACCTGTGCTGG GTGCGTAGCGGCATCTCGCGGGCTGCGCTG CTGCGCCTTTTCTTGGCCATGGAGCGTGGT AGCCACTTCAGCCTGGGCTGTCCGCAGCTG GGCTACGCCGCGGCCCGTGCCTTCCGCCTA GAGCCGCTCACACCACGCGGCGTGCTCACA GTGGACGGGGAGCAGGTGGAGTATGGGCCG CTACAGGCACAGATGCACCCTGGCATCGGT ACACTGCTCACTGGGCCTCCTGGCTGCCCG GGGCGGGAGCCCTGA (SEQ ID NO: 18) CerK ATGGGGGCGACGGGGGCGGCGGAGCCGCTG CAATCCGTGCTGTGGGTGAAGCAGCAGCGC TGCGCCGTGAGCCTGGAGCCCGCGCGGGCT CTGCTGCGCTGGTGGCGGAGCCCGGGGCCC GGAGCCGGCGCCCCCGGCGCGGATGCCTGC TCTGTGCCTGTATCTGAGATCATCGCCGTT GAGGAAACAGACGTTCACGGGAAACATCAA GGCAGTGGAAAATGGCAGAAAATGGAAAAG CCTTACGCTTTTACAGTTCACTGTGTAAAG AGAGCACGACGGCACCGCTGGAAGTGGGCG CAGGTGACTTTCTGGTGTCCAGAGGAGCAG CTGTGTCACTTGTGGCTGCAGACCCTGCGG GAGATGCTGGAGAAGCTGACGTCCAGACCA AAGCATTTACTGGTATTTATCAACCCGTTT GGAGGAAAAGGACAAGGCAAGCGGATATAT GAAAGAAAAGTGGCACCACTGTTCACCTTA GCCTCCATCACCACTGACATCATCGTTACT GAACATGCTAATCAGGCCAAGGAGACTCTG TATGAGATTAACATAGACAAATACGACGGC ATCGTCTGTGTCGGCGGAGATGGTATGTTC AGCGAGGTGCTGCACGGTCTGATTGGGAGG ACGCAGAGGAGCGCCGGGGTCGACCAGAAC CACCCCCGGGCTGTGCTGGTCCCCAGTAGC CTCCGGATTGGAATCATTCCCGCAGGGTCA ACGGACTGCGTGTGTTACTCCACCGTGGGC ACCAGCGACGCAGAAACCTCGGCGCTGCAT ATCGTTGTTGGGGACTCGCTGGCCATGGAT GTGTCCTCAGTCCACCACAACAGCACACTC CTTCGCTACTCCGTGTCCCTGCTGGGCTAC GGCTTCTACGGGGACATCATCAAGGACAGT GAGAAGAAACGGTGGTTGGGTCTTGCCAGA TACGACTTTTCAGGTTTAAAGACCTTCCTC TCCCACCACTGCTATGAAGGGACAGTGTCC TTCCTCCCTGCACAACACACGGTGGGATCT CCAAGGGATAGGAAGCCCTGCCGGGCAGGA TGCTTTGTTTGCAGGCAAAGCAAGCAGCAG CTGGAGGAGGAGCAGAAGAAAGCACTGTAT GGTTTGGAAGCTGCGGAGGACGTGGAGGAG TGGCAAGTCGTCTGTGGGAAGTTTCTGGCC ATCAATGCCACAAACATGTCCTGTGCTTGT CGCCGGAGCCCCAGGGGCCTCTCCCCGGCT GCCCACTTGGGAGACGGGTCTTCTGACCTC ATCCTCATCCGGAAATGCTCCAGGTTCAAT TTTCTGAGATTTCTCATCAGGCACACCAAC CAGCAGGACCAGTTTGACTTCACTTTTGTT GAAGTTTATCGCGTCAAGAAATTCCAGTTT ACGTCGAAGCACATGGAGGATGAGGACAGC GACCTCAAGGAGGGGGGGAAGAAGCGCTTT GGGCACATTTGCAGCAGCCACCCCTCCTGC TGCTGCACCGTCTCCAACAGCTCCTGGAAC TGCGACGGGGAGGTCCTGCACAGCCCTGCC ATCGAGGTCAGAGTCCACTGCCAGCTGGTT CGACTCTTTGCACGAGGAATTGAAGAGAAT CCGAAGCCAGACTCACACAGCTGA (SEQ ID NO: 19)

EXAMPLES

Mice

[0067] All animal procedures were performed under protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Care and Use Committee.

Synthesis of Anc80.A C

[0068] 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.

Anc80 Plasmid Sequence

[0069]

TABLE-US-00002 pAAV.CMV. CTGCGCGCTCGCTCGCTCACTGAGGCCGCC WPRE.bGH.d CGGGCAAAGCCCGGGCGTCGGGCGACCTTT na GGTCGCCCGGCCTCAGTGAGCGAGCGAGCG CGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTTGTAGTTAATGATTAACCCG CCATGCTACTTATCTACGTAGCCATGCTCT AGGAAGATCGGAATTCGCCCTTAAGCTAGC TAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCG CGTTACATAACTTACGGTAAATGGCCCGCC TGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGT AACGCCAATAGGGACTTTCCATTGACGTCA ATGGGTGGAGTATTTACGGTAAACTGCCCA CTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGG TAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCA GTACATCTACGTATTAGTCATCGCTATTAC CATGGTGATGCGGTTTTGGCAGTACATCAA TGGGCGTGGATAGCGGTTTGACTCACGGGG ATTTCCAAGTCTCCACCCCATTGACGTCAA TGGGAGTTTGTTTTGGCACCAAAATCAACG GGACTTTCCAAAATGTCGTAACAACTCCGC CCCATTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCTGG TTTAGTGAACCGTCAGATCCTGCAGAAGTT GGTCGTGAGGCACTGGGCAGGTAAGTATCA AGGTTACAAGACAGGTTTAAGGAGACCAAT AGAAACTGGGCTTGTCGAGACAGAGAAGAC TCTTGCGTTTCTGATAGGCACCTATTGGTC TTACTGACATCCACTTTGCCTTTCTCTCCA CAGGTGTCCAGGCGGCCGCNNNGGATCCAA TCAACCTCTGGATTACAAAATTTGTGAAAG ATTGACTGGTATTCTTAACTATGTTGCTCC TTTTACGCTATGTGGATACGCTGCTTTAAT GCCTTTGTATCATGCTATTGCTTCCCGTAT GGCTTTCATTTTCTCCTCCTTGTATAAATC CTGGTTGCTGTCTCTTTATGAGGAGTTGTG GCCCGTTGTCAGGCAACGTGGCGTGGTGTG CACTGTGTTTGCTGACGCAACCCCCACTGG TTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTAT TGCCACGGCGGAACTCATCGCCGCCTGCCT TGCCCGCTGCTGGACAGGGGCTCGGCTGTT GGGCACTGACAATTCCGTGGTGTTGTCGGG GAAATCATCGTCCTTTCCTTGGCTGCTCGC CTGTGTTGCCACCTGGATTCTGCGCGGGAC GTCCTTCTGCTACGTCCCTTCGGCCCTCAA TCCAGCGGACCTTCCTTCCCGCGGCCTGCT GCCGGCTCTGCGGCCTCTTCCGCGTCTTCG AGATCTGCCTCGACTGTGCCTTCTAGTTGC CAGCCATCTGTTGTTTGCCCCTCCCCCGTG CCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATT GCATCGCATTGTCTGAGTAGGTGTCATTCT ATTCTGGGGGGTGGGGTGGGGCAGGACAGC AAGGGGGAGGATTGGGAAGACAATAGCAGG CATGCTGGGGACTCGAGTTAAGGGCGAATT CCCGATAAGGATCTTCCTAGAGCATGGCTA CGTAGATAAGTAGCATGGCGGGTTAATCAT TAACTACAAGGAACCCCTAGTGATGGAGTT GGCCACTCCCTCTCTGCGCGCTCGCTCGCT CACTGAGGCCGGGCGACCAAAGGTCGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGCCTTAATTAACC TAATTCACTGGCCGTCGTTTTACAACGTCG TGACTGGGAAAACCCTGGCGTTACCCAACT TAATCGCCTTGCAGCACATCCCCCTTTCGC CAGCTGGCGTAATAGCGAAGAGGCCCGCAC CGATCGCCCTTCCCAACAGTTGCGCAGCCT GAATGGCGAATGGGACGCGCCCTGTAGCGG CGCATTAAGCGCGGCGGGTGTGGTGGTTAC GCGCAGCGTGACCGCTACACTTGCCAGCGC CCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCC CCGTCAAGCTCTAAATCGGGGGCTCCCTTT AGGGTTCCGATTTAGTGCTTTACGGCACCT CGACCCCAAAAAACTTGATTAGGGTGATGG TTCACGTAGTGGGCCATCGCCCTGATAGAC GGTTTTTCGCCCTTTGACGTTGGAGTCCAC GTTCTTTAATAGTGGACTCTTGTTCCAAAC TGGAACAACACTCAACCCTATCTCGGTCTA TTCTTTTGATTTATAAGGGATTTTGCCGAT TTCGGCCTATTGGTTAAAAAATGAGCTGAT TTAACAAAAATTTAACGCGAATTTTAACAA AATATTAACGTTTATAATTTCAGGTGGCAT CTTTCGGGGAAATGTGCGCGGAACCCCTAT TTGTTTATTTTTCTAAATACATTCAAATAT GTATCCGCTCATGAGACAATAACCCTGATA AATGCTTCAATAATATTGAAAAAGGAAGAG TATGAGTATTCAACATTTCCGTGTCGCCCT TATTCCCTTTTTTGCGGCATTTTGCCTTCC TGTTTTTGCTCACCCAGAAACGCTGGTGAA AGTAAAAGATGCTGAAGATCAGTTGGGTGC ACGAGTGGGTTACATCGAACTGGATCTCAA TAGTGGTAAGATCCTTGAGAGTTTTCGCCC CGAAGAACGTTTTCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATC CCGTATTGACGCCGGGCAAGAGCAACTCGG TCGCCGCATACACTATTCTCAGAATGACTT GGTTGAGTACTCACCAGTCACAGAAAAGCA TCTTACGGATGGCATGACAGTAAGAGAATT ATGCAGTGCTGCCATAACCATGAGTGATAA CACTGCGGCCAACTTACTTCTGACAACGAT CGGAGGACCGAAGGAGCTAACCGCTTTTTT GCACAACATGGGGGATCATGTAACTCGCCT TGATCGTTGGGAACCGGAGCTGAATGAAGC CATACCAAACGACGAGCGTGACACCACGAT GCCTGTAGTAATGGTAACAACGTTGCGCAA ACTATTAACTGGCGAACTACTTACTCTAGC TTCCCGGCAACAATTAATAGACTGGATGGA GGCGGATAAAGTTGCAGGACCACTTCTGCG CTCGGCCCTTCCGGCTGGCTGGTTTATTGC TGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTA CACGACGGGGAGTCAGGCAACTATGGATGA ACGAAATAGACAGATCGCTGAGATAGGTGC CTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGA TTTAAAACTTCATTTTTAATTTAAAAGGAT CTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGAT CAAAGGATCTTCTTGAGATCCTTTTTTTCT GCGCGTAATCTGCTGCTTGCAAACAAAAAA ACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAA GGTAACTGGCTTCAGCAGAGCGCAGATACC AAATACTGTCCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACC GCCTACATACCTCGCTCTGCTAATCCTGTT ACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATA GTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTT GGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCAC GCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGA GCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCG CCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAA AAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGCGGTTTTGCTCACAT GTTCTTTCCTGCGTTATCCCCTGATTCTGT GGATAACCGTATTACCGCCTTTGAGTGAGC TGATACCGCTCGCCGCAGCCGAACGACCGA GCGCAGCGAGTCAGTGAGCGAGGAAGCGGA AGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTG GCACGACAGGTTTCCCGACTGGAAAGCGGG CAGTGAGCGCAACGCAATTAATGTGAGTTA GCTCACTCATTAGGCACCCCAGGCTTTACA CTTTATGCTTCCGGCTCGTATGTTGTGTGG AATTGTGAGCGGATAACAATTTCACACAGG AAACAGCTATGACCATGATTACGCCAGATT TAATTAAGG (SEQ ID NO: 20)

[0070] 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.

TABLE-US-00003 TABLE 2 SEQ SEQ ID ID Gene Forward NO. Reverse NO. AC ACAGGATTCA 21 TGGGCATCTT 22 AACCAGGACT TCCTTCCGAA GT AC TGACAGGATT 23 CTGGGCATCT 24 CAAACCAGGA TTCCTTCCGA CT Sphk1 ATACTCACCG 25 CCATTAGCCC 26 AACGGAAGAA ATTCACCACC CC TC Sphk1 ACTGATACTC 27 CATTAGCCCA 28 ACCGAACGGA TTCACCACCT A C S1PR2 CACAGCCAAC 29 TCTGAGTATA 30 AGTCTCCAAA AGCCGCCCA S1PR2 ATAGACCGAG 31 GAACCTTCTC 32 CACAGCCAA AGGATTGAGG T 18s rRNA* TAACGAACGA 33 CGGACATCTA 34 GACTCTGGCA AGGGCATCAC T AG *Genetic Vaccines and Therapy 2004, 2:5

Western Blot

[0071] Upon thawing, hearts lysates' were subjected to separation by SDS-PAGE using 12% precast Nupage Bis/Tris gels (Invitrogen, Carlsbad, Calif., 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, Ill.). For molecular weight determination prestained protein standards (Amersham, Buckinghamshire, UK) were used.

Immunohistochemistry

[0072] The mouse hearts were harvested and perfused using perfusion buffer (2 g/I butanedione, monoxime and 7.4 g/I KCI 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 μM 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.

[0073] Model of PAH A rat PAH model was used. Pneumonectomy combined with Sugen rat model results in fast pulmonary vascular remodeling comparable to clinical PAH and development of the plexiform lesions found in human PAH. AC gene was introduced using Anc80 as viral vector to the lung via intratracheal transfer.

Cardiovascular Evaluation

[0074] MRI was used to assess the effect of Anc80-AC on heart function and PAH parameters (right ventricular hemodynamics including ejection fraction, hypertrophy, pulmonary artery pressure and vascular resistance).

Tissue Evaluation

[0075] Animal tissues from Sprague-Dawley rats will be analyzed for RNA sequencing, proteomics and sphingolipids quantification.

[0076] Study groups: 1. No Anc80/AC no PAH; 2. Saline +PAH; 3. Anc80 only+PAH; 4. Anc80/AC, No PAH; 5. Anc80/AC+PAH.

Preliminary Results

[0077] Rats were subjected to PAH induction protocol (FIG. 2). At week 0, rats were subjected to baseline MRI, RV and PA catheterization to measure the pressure, followed by left lung removal. On day 7 pneumonectomized rats were subjected to SU5416 (Su/Pn 10 mg/kg) administration (SC injection). Induced animals demonstrated severely elevated mean PA pressures and developed neointima and smooth muscle hypertrophy (FIGS. 3 and 4). On week 4 PAH induced rats were treated with Anc80 AC (1×10.sup.11genome copies). On weeks 6 and 8 animals were validated by MRI for heart function and RV and PA catheterization for pressure measurement.

[0078] Preliminary PAH results with AC-Anc80 gene therapy were outstanding (see FIGS. 5-10). In control animals a severe PAH develops after SU5416 (Su/Pn) administration to pneumonectomized rats. Induced animals demonstrated severely elevated mean PA pressures and developed neointima and smooth muscle hypertrophy. After AC administration cardiac output increased in 32% (FIG. 5), right ventricular systolic volume decreased in 39% (FIG. 6), right ventricular ejection fraction increased in 65% (FIG. 7), mean pulmonary artery pressure decreased in 94% (FIG. 8) and mean pulmonary vascular resistance decreased in 4.8 times (FIG. 9). Animals treated with AC Anc80 at 8 weeks showed excellent cardiac function (validated by MRI, FIG. 10) and normal PA pressures despite PAH disease present. In one embodiment, ancestral 80 (Anc80) viral vector was used for gene delivery. Anc80 has been used as a viral vector with low immunogenicity and ancestral strains that are not regularly recognized by human antibodies, unlike common AAVs that are seropositive in >50% of the population. Anc80 delivery has provided rapid onset of expression in less than 48 hours. The Anc80 virus demonstrated the ability to generate very high transduction of lung and other cardiovascular tissues. Overall, the Anc80 viral vector provided an ideal delivery vehicle for the gene (FIG. 17).

[0079] 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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