Compositions and methods for gene therapy

Abstract

A viral vector includes a cardiomyocyte-specific promoter and a polynucleotide operably linked to the cardiomyocyte-specific promoter. The polynucleotide encodes an enzyme that mediates expression of one or more specialized pro-resolving mediators. The viral vector may be formulated into a pharmaceutical composition. The pharmaceutical composition may be administered to subject having or at risk of having a condition treatable with the viral vector.

Description

[0001] PCT Patent Application

[0002] Attorney Docket No. 0110.000765W001

[0003] COMPOSITIONS AND METHODS FOR GENE THERAPY

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 718,065, filed November 8, 2024, which is incorporated herein by reference in its entirety.

[0006] GOVERNMENT FUNDING

[0007] This invention was made with government support under HL165704 awarded by the National institutes of Health. The government has certain rights in the invention.

[0008] SUMMARY

[0009] This disclosure describes, in one aspect, a viral vector that includes a cardiomyocytespecific promoter and a polynucleotide operably linked to the cardiomyocyte-specific promoter. The polynucleotide encodes an enzyme that mediates expression of one or more specialized proresolving mediators.

[0010] In one or more embodiments, the cardiomyocyte-specific promoter is an a-myosin heavy chain (u-MHC) promoter. In one or more embodiments, the cardiomyocyte-specific promoter is a troponin T promoter.

[0011] In one or more embodiments, the viral vector is an adeno-associated virus serotype 1 (AAV1). In one or more embodiments, the viral vector is an adeno-associated virus serotype 2 (AAV2). In one or more embodiments, the viral vector is an adeno-associated virus serotype 9 (AAV9).

[0012] In one or more embodiments, the enzyme that mediates expression of one or more specialized pro-resolving mediators is arachidonate 5-lipoxygenase (5-LOX).

[0013] In another aspect, this disclosure describes a pharmaceutical composition that includes any embodiment of the viral vector encompassed by this disclosure and a pharmaceutically acceptable carrier.

[0014] In another aspect, this disclosure describes a method of overexpressing 5-LOX in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure having a polynucleotide that encodes arachidonate 5 -lipoxygenase (5-LOX).

[0015] In another aspect, this disclosure describes a method of increasing expression of a proresolving mediator (SPM) in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0016] In one or more embodiments, the SPM is resolvin DI (RvDl).

[0017] In another aspect, this disclosure describes a method of reducing NLRP3 inflammasome expression in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0018] In another aspect, this disclosure describes a method of reducing inflammation in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0019] In another aspect, this disclosure describes a method of increasing inflammation resolution in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0020] In another aspect, this disclosure describes a method of treating heart failure with preserved ejection fraction (HFpEF) in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0021] In another aspect, this disclosure describes a method of reducing oxidative modification of cMyBPC S-glutathionylation in a subject. Generally, the method includes administering to the subject any embodiment of the pharmaceutical composition encompassed by the present disclosure.

[0022] In one or more embodiments, the subject has diabetes mellitus.

[0023] In one or more embodiments, the subject has a high fat diet.

[0024] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1. DM reduces cardiac RvDl and 5-LOX. (A) Fold change of RvDl as measured by ELISA assay and (B) 5-LOX as measured by immunoblotting were significantly reduced in HFD-induced DM hearts. Representative immunoblotting images of 5-LOX are shown. (C) E / E', an echocardiographic indicator of cardiac diastolic function, was improved by intraperitoneal injection of RvDl at 200 ng / mouse for 2 weeks. N = 7-12 per group. Bars are mean ± SEM. Unpaired t-test was used. *P<0.05; ***P<0.001. Ctrl, control; DM, diabetes mellitus; E / E', the ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annual velocity; HFD, high fat diet; 5-LOX, 5-lipoxygenase; RvDl, resolvin DI .

[0027] FIG. 2. Cardiac specific 5-LOX overexpression improves DM-associated diastolic dysfunction (DD). (A) Cardiac 5-LOX level by immunoblotting was increased in AAV9-A / ox5 (5-LOX gene) treated DM mice. Representative immunoblotting images are shown. (B) Cardiac RvDl by ELISA assay was enhanced by cardiac 5-LOX overexpression. (C) Cardiac specific 5- LOX overexpression improved DM-associated DD by echocardiography. (D) NLRP3 expression was reduced by cardiac 5-LOX overexpression. Representative immunoblotting images are shown. (E) Fasting glucose level was not affected by cardiac 5-LOX upregulation. (F) Body weight was not affected by cardiac 5-LOX upregulation. N= 6-7 mice per group. Bars are mean ± SEM. Unpaired t-test was used. *P<0.05; **P<0.01. AAV9, adeno-associated virus serotype 9; DM, diabetes mellitus; E / E', the ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annual velocity; 5-LOX, 5-lipoxygenase; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; RvDl, resolvin DI.

[0028] FIG. 3. Cardiac macrophage number and inflammatory properties are unaltered by cardiac specific 5-LOX overexpression. (A) Cardiac MCP-1, (B) CD68 as measured by immunoblotting, and (C) IL- 10 level as measured by ELISA assay were comparable between the AAV9-plain and XXN9-Alox5 vector treated DM mice. N = 6-7 mice per group. Bars are mean ± SEM. Unpaired t-test was used. AAV9, adeno-associated virus serotype 9; DM, diabetes mellitus; IL-10, interleukin-1 beta; MCP-1, monocyte chemoattractant protein-1. FIG. 4. Cardiac RvDl and 5-LOX are reduced in DM-associated HFpEF. (A) Cardiac 5- LOX level measured by immunoblotting was significantly reduced in HFD-induced DM hearts. (B) Cardiac RvDl level measured by ELISA assay was significantly reduced in HFD-induced DM hearts. N=7-12 mice per group. Bars are mean ± SEM. Unpaired t-test was used. *P<0.05; ***p<0.001. Ctrl, control; DM, diabetes mellitus; HFD, high fat diet; HFpEF, heart failure with preserved ejection fraction; 5-LOX, 5 -lipoxygenase; RvDl, resolvin DI.

[0029] FIG. 5. Cardiomyocyte overexpression of 5-LOX improved diastolic function. (A) AAV9M / ox5 treated DM hearts showed enhanced cardiac 5-LOX level by immunobloting. (B) AA 9-A / ox5 treated DM hearts showed improved E / E' (an echocardiographic indicator of cardiac diastolic function). (C) Immunoblot data showing AAV9M / ox5 treated DM hearts had reduced cardiac S-glutathionylated MyBPC level that was elevated in AAV9-plain vector treated DM mice. (D) Ejection fraction was unchanged by AAV9- / l / x5 treatment. (E) Fasting glucose was unchanged by AAV9-Alox5 treatment. (F) Body weight was unchanged by AAM9-Alox5 treatment. N=5-l 1 mice per group. Bars are mean ± SEM. Unpaired t-test (A) or 1-way analysis of variance with Bonferroni post hoc tests (B-F) was used. *P<0.05; **P<0.01, ***P<0.001. AAV9, adeno-associated virus serotype 9; Ctrl, control; DM, diabetes mellitus; E / E', the ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annual velocity; EF, ejection fraction; Alox5, arachidonate 5 -Lipoxygenase gene encoding 5-LOX; MyBPC, myosin binding protein C; S-glu, S-glutathionylation.

[0030] FIG. 6. Cardiomyocyte specific overexpression of 5-LOX raised RvDl levels without affecting LTA4. (A) Cardiac RvDl level measured by ELISA was significantly increased in AAN9-Alox5 treated DM hearts. (B) Leukotriene A4 measured by ELISA was unchanged in AAA9-Alox5 treated DM hearts. N = 6 mice per group. Bars are mean ± SEM. Unpaired t-test was used. *P<0.05. AAV9, adeno-associated virus serotype 9; Alox5, arachidonate 5- Lipoxygenase gene; DM, diabetes mellitus; 5-LOX, 5 -lipoxygenase; NS, not significant; LTA4, leukotriene A4; RvDl, resolvin DI.

[0031] FIG. 7. RvDl treatment improved diastolic function in a similar manner to cardiomyocyte 5-LOX overexpression. (A) Intraperitoneal injection of RvDl at 100 ng / mouse for 2-3 weeks had a similar effect on E / E' as did cardiomyocyte 5-LOX overexpression. (B) Intraperitoneal injection of RvDl at 100 ng / mouse for 2-3 weeks did not affect EF. N = 6-7 mice per group. Bars are mean ± SEM. One-way analysis of variance with Bonferroni post hoc tests was used. **P<0.01, ***P<0.001 . Ctrl, control; DM, diabetes mellitus; E / E', the ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annual velocity; EF, ejection fraction; RvDl, resolvin DI. Control and DM values were measured contemporaneously and are reproduced from FIG. 5 for comparison.

[0032] FIG. 8. Cardiac specific 5-LOX overexpression did not alter cardiac macrophages. (A) Immunoblot data showing cardiac CD68 was significantly higher in DM mice and was unaltered regardless either AAV9-plain or AAV9-d / <->x5 vector treatment. (B) Immunoblot data showing IL-ip was significantly higher in DM mice and was unaltered regardless either AAV9-plain or AAV9-A / ox5 vector treatment. Representative immunoblotting images are shown. N = 5-7 mice per group. Bars are mean ± SEM. One-way analysis of variance with Bonferroni post hoc tests were used was used. **P<0.01, ***P<0.001. AAV9, adeno-associated virus serotype 9; Alox5, arachidonate 5 -Lipoxygenase gene; Ctrl, control; DM, diabetes mellitus; IL-ip, interleukin-ip.

[0033] FIG. 9. Summary of how cardiomyocyte-specific 5-LOX overexpression improves DM- associated HFpEF. AAV9, adeno-associated virus serotype 9; Alox5, arachidonate 5- lipoxygenase gene; CM, cardiomyocytes, DM, diabetes mellitus; E / E', the ratio of mitral inflow velocity E and longitudinal tissue velocity of the mitral anterior annulus E'; MO, macrophages, S-glu-cMyBPC, S-glutathionylated cardiac myosin binding protein C; RvDl, resolvin DI .

[0034] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] This disclosure describes cardiac-specific gene therapy compositions and methods. Generally, the method includes overexpressing genes that enhance resolution of inflammation signaling or production of specialized pro-resolving molecules or reducing the expression of genes that signal or produce inflammatory molecular species such as polyunsaturated fatty acid derivatives prostaglandins, thromboxanes, and leukotrienes. In one or more embodiments, the compositions and methods described herein may be employed in the treatment of heart failure with preserved ejection fraction (HFpEF).

[0036] Heart failure (HF) is a health concern in which many cases are characterized by normal ejection fraction and impaired diastolic relaxation of myocardium, known as heart failure with preserved ejection fraction (HFpEF). With a similar mortality to heart failure with reduced ejection fraction (HFrEF), there are limited specific therapies because of a lack of knowledge on the pathogenesis mechanism of the disease. Many HFpEF patients have co-existing diabetes mellitus (DM), and DM has a significantly negative impact on the HFpEF prognosis. Diabetes mellitus causes macrophage- mediated cardiac inflammation, which can lead to heart failure with preserved ejection fraction (HFpEF).

[0037] A subset of lipid-derived signaling molecules known as specialized pro-resolving mediators (SPMs) can promote inflammation resolution, and SPM deficit leads to chronic inflammation. SPMs are derived from essential fatty acids under the action of lipoxygenases. Arachidonate 5 -lipoxygenase (5-LOX), encoded by the AL0X5 gene, is a lipoxygenase that is highly expressed in leukocytes and biosynthesizes specialized pro-resolving mediators (SPMs), such as lipoxins and resolvins. For example, resolvin DI (RvDl) is the product of 5-LOX from docosahexaenoic acid (DHA). 5-LOX can inhibit excessive inflammation, promote leukocyte clearance, and / or promote resolution of inflammation in myocardial infarcts. Similarly, RvDl contributes to the attenuation of cardiac inflammation and cardiac remodeling in the settings of various heart diseases.

[0038] This disclosure describes inducing upregulation of 5-LOX and / or its product RvDl to improve DM-associated HFpEF. While 5-LOX is predominantly expressed in myeloid leukocytes such as neutrophils, macrophages, and dendritic cells, this disclosure describes using a cardiomyocyte-specific approach to avoid the off-target effects on systemic immunity.

[0039] As described in more detail below, diabetes mellitus was induced by feeding mice with a high fat diet (HFD) for 20-24 weeks. To reduce off-target immunomodulation, 5-LOX was upregulated specifically in cardiomyocytes of DM mice via intravenous delivery of the adeno- associated viral vectors (serotype 9, AAV9) with the cardiac specific promoter a-myosin heavy chain (a-MHC). Another DM cohort underwent intraperitoneal RvDl injection (100 ng / mouse) at 24 weeks old for two weeks. Echocardiography was performed at the age of 26-30 weeks.

[0040] DM mice showed lower cardiac RvD l levels and 5-LOX expression than control mice. Both RvDl treatment and cardiac specific 5-LOX upregulation improved DM-associated diastolic dysfunction (DD). Cardiac specific 5-LOX gene therapy also significantly enhanced 5- LOX expression, enhanced RvDl levels, and reduced NLRP3 inflammasome expression in DM hearts without affecting glucose metabolism or obesity. Cardiac macrophage infiltration and interleukin- 1 beta (IL- 1 ) secretion were unchanged by cardiac 5-LOX upregulation. Both direct RvDl treatment and cardiac-specific gene therapy enhanced resolution of inflammation in cardiomyocytes and reversed DM-associated HFpEF. Moreover, cardiacspecific gene therapy avoided off-target immunosuppression. Therefore, this disclosure describes AAV-directed cardiac 5-LOX upregulation as a novel cardiac-specific gene therapy for HFpEF.

[0041] Diabetes mellitus decreased cardiac RvDl and 5-LOX levels

[0042] Diabetes mellitus-associated diastolic dysfunction (DD) is related to unresolved inflammation, and inflammation resolution is promoted by specialized pro-resolving mediators (SPMs). Here the cardiac level of RvDl, an SPM produced by docosahexaenoic acid (DHA) under the action of 5-LOX, was investigated. RvDl was significantly lower in DM hearts compared to the control hearts (FIG. 1A, 1.00 ± 0.07-fold change in control versus 0.53 ± 0.04- fold change in DM, P<0.0001; FIG. 4B, DM: 0.53 ± 0.04-fold of control, P<0.001). 5-LOX, the synthetic enzyme that generates RvDl, was also significantly reduced in the DM hearts (FIG. IB, 1.01 ± 0.12-fold change in control versus 0.72 ± 0.05-fold change in DM, P=0.046; FIG. 4A, DM: 0.72 ± 0.05-fold of control, P=0.046). Supplementing RvDl by intraperitoneal injection improved DM-associated diastolic dysfunction (FIG. 1C, E / E', 22.9 ± 0.4-fold change in DM versus 19.7 ± 1.4-fold change in DM+RvDl, P=0.045).

[0043] These data suggest that inflammation resolution was impaired in DM hearts and that promoting inflammation resolution ameliorated DM-associated diastolic dysfunction.

[0044] Cardiac specific 5-LOX overexpression improved DM-associated diastolic dysfunction

[0045] 5-LOX is involved in cardiac inflammation resolution and myocardial repair, is mainly expressed in leukocytes, and regulates the synthesis of many SPMs, such as RvDl. To correct the cardiac 5-LOX level while limiting the off-target effects of 5-LOX regulation on leukocytes, the Alox5 gene was specifically overexpressed in cardiomyocytes using an adeno-associated viral (serotype 9, AAV9) approach with the Alox5 gene driven by a cardiomyocyte specific promoter, a-myosin heavy chain (a-MHC).

[0046] As shown in FIG. 2A and FIG. 5A, AAV9-A / o 5 vectors successfully restored 5-LOX expression in DM hearts (FIG. 2A: 0.70 ± 0.04-fold change in DM+AAV9-plain versus 0.89 ± 0.06-fold change in DM+AAV9-Afox5, P=0.020; FIG. 5A: DM+AAV9Mfox5: 1.27 ± 0.08-fold of control, P=0.031). Consequently, the cardiac RvDl level was almost twice that of the DM mice receiving AAV9-plain vectors (FIG. 2B, 1.25 ± 0.19-fold change in DM + AAV9-plain versus 2.11 ± 0.31-fold change in DM + AAV9- / 4 / ox5, P=0.038). The increase of cardiac 5-LOX and RvDl levels was accompanied by an improvement in cardiac diastolic function. The E / E' ratio was lower in 5-LOX overexpressing DM hearts (FIG. 2C, 20.5 ± 1.0-fold change in DM + AAV9-plain versus 17.0 ± 1.2-fold change in DM + AAV9-A / ox5, P=0.045; Figure 5B, 24.4 ± 1.3 in DM+AAV9-plain vs. 20.3 ± 0.5 in DM+AAV9-^fo 5, P=0.013).

[0047] To examine the cardiac inflammation after 5-LOX overexpression, the expression of the NLRP3 inflammasome, a common inflammatory marker, was investigated. Cardiac 5-LOX upregulation significantly decreased the NLRP3 level in DM hearts (FIG. 2D, 0.90 ± 0.02 in DM+AAV9-plain versus 0.80 ± 0.02 in DM+AAV9-^4 / ox5, P=0.002), suggesting alleviated cardiac inflammation.

[0048] Increased cardiomyocyte mitochondrial reactive oxygen species (ROS) leads to increased S-glutathionylation of cardiac myosin binding protein C (cMyBPC), which is causative of DD and is also elevated in the serum of humans with HFpEF. Cardiac specific 5-LOX upregulation normalized S-glutathionylation of cMyBPC (FIG. 5C, 1.42 ± 0.10-fold of control in DM+AAV9- plain vs. 0.88 ± 0.10-fold of control in DM+AAV9-Alox5, P=0.002), suggesting that 5-LOX overexpression improved DM-associated DD by reducing myocardial oxidative stress.

[0049] There were no apparent changes in ejection fraction value (FIG. 5D), fasting glucose level (FIG. 2E and FIG. 5E) or body weight (FIG. 2F and FIG. 5F) after cardiac 5-LOX upregulation. These data indicate that cardiomyocyte-specific 5-LOX upregulation reduces cardiac inflammation, promote inflammation resolution, and / or ameliorate DM-associated diastolic dysfunction.

[0050] 5-LOX overexpression improved cardiac RvDl (FIG. 6A, 1.69 ± 0.25-fold of DM+AAV9-plain, P=0.038). 5-LOX also participates in the synthesis of inflammatory leukotrienes, especially in immune cells. Nevertheless, by overexpressing 5-LOX specifically in cardiomyocytes, leukotriene A4 (LTA4) was unchanged in the heart (FIG. 6B, 250.3 ± 11.2 pg / mg in DM+AAV9-plain vs. 281.3 ± 15.6 pg / mg in DM+AAV9-Alox5, P=0.128). These data indicated that cardiomyocyte-specific 5-LOX upregulation improved DD and enhanced SPMs without increasing inflammatory markers.

[0051] RvDl supplement improved DD in a similar manner to 5-LOX overexpression To confirm that RvDl can alter DM-associated DD, RvDl was systemically administered via intraperitoneal injection to DM mice for two-to-three weeks. This treatment with RvDl had an analogous effect to cardiac specific 5-LOX overexpression on improving diastolic function (FIG. 7 A, E / E', 26.2 ± 1.5 in DM vs. 19.7 ± 1.4 in DM+RvDl, P<0.01) without affecting systolic function (FIG. 7B).

[0052] Cardiac specific 5-LOX overexpression caused no impact on cardiac macrophages Pro-inflammatory macrophages accumulate in DM hearts, which can lead to DM- associated diastolic dysfunction through interleukin- 1 beta (IL-10) secretion. Here, although diastolic dysfunction was improved by cardiac 5-LOX overexpression, cardiac macrophage infiltration was not reduced in DM hearts. Cardiac MCP-1, a chemokine that regulates monocytes / macrophage migration and infiltration, was unaltered in A AV9-4 / x5 vector treated DM mice (FIG. 3A, 0.49 ± 0.02 in DM+AAV9-plain versus 0.50 ± 0.01 in DM+AAV9-d / x5, P=0.639). The expression of macrophage marker, CD68, was comparable between the two groups (FIG. 3B, 0.32 ± 0.01 in DM+AAV9-plain versus 0.33 ± 0.02 in DM+AAV9M / ox5, P=0.723; FIG. 8A, 1.54 ± 0.09-fold of control in DM+AAV9 -plain, 1.47 ± 0.08-fold of control in DM+AAV9M / ox5).

[0053] Macrophages are one source of cardiac IL- 10. Consistent with increased macrophage infiltration in DM hearts, cardiac IL- 10 level was significantly higher in DM than control hearts (FIG. 8B, DM: 1.53 ± 0.05-fold of control, P<0.001). In DM hearts that overexpress 5-LOX, the IL-10 level remained unchanged compared to the control AAV9 treated DM hearts (FIG. 3C, 20.5 ± 0.47 pg / mL in DM+AAV9-plain versus 20.2 ± 0.58 pg / mL in DM+AAV9-.4 / \o, P=0.699; FIG. 8B, DM+AAV9-plain: 1.59 ± 0.06-fold of control vs. 1.45 ± 0.09-fold of control in DM+AAV9M / ox5, P=0.225). These results are consistent with macrophages, not cardiomyocytes, being the predominant source of IL- 10, and alterations in cardiomyocyte IL- 10 production having limited influence on total cardiac IL- 10 despite the cardiomyocytes being the end operator of diastolic relaxation. Although not having measured direct macrophage subtypes by cell surface markers, the lack of change in total IL- 10 and macrophages as measured by CD68 suggests that alterations in cardiac macrophage phenotype were not responsible for the improvement in diastolic dysfunction and implied that upregulating 5-LOX specifically in cardiomyocytes had no effect on cardiac macrophage number or their pro-inflammatory properties.

[0054] Thus, this disclosure shows that DM-associated diastolic dysfunction is characterized by reduced levels of the specialized pro-resolving mediator RvDl that results from reduced levels of 5-LOX, which is responsible for the synthesis of RvDl. The pathogenic role of this reduced level of RvDl was reinforced by the observation that RvDl supplementation improved diastolic dysfunction.

[0055] To achieve a cardiac specific reduction in inflammation without systemic consequences, the gene for 5-LOX, the protein responsible for the synthesis of RvDl, was overexpressed in heart using the cardiac specific promoter, a-MHC. 5-LOX has pro-resolving effects in the heart, but it is not generally expressed in cardiomyocytes. Nevertheless, cardiac overexpression reduced inflammation, cMyBPC oxidative modification, and concomitant diastolic dysfunction without altering the inflammatory macrophage phenotype seen in HFpEF or cardiac macrophage burden. 5-LOX overexpression increased the specialized pro-resolving mediator RvDl without affecting cardiac leukotrienes level and, since 5-LOX and RvDl have similar effects on diastolic dysfunction, it is likely that 5-LOX overexpression acted through increasing RvDl.

[0056] Macrophage production of IL-ip, along with reactive oxygen species (ROS) production, mediates the development of impaired diastolic function secondary to DM-induced inflammation. Accordingly, inhibiting macrophage secretion of IL-ip or mitochondrial ROS scavenging are sufficient to reverse DM-induced diastolic dysfunction. This disclosure shows that the overexpression of 5-LOX in cardiomyocytes reduced inflammation and ROS without exerting any effect on macrophage chemotaxis or IL-ip signaling. These findings suggest that the proposed macrophage-cardiomyocyte crosstalk may be a one-way communication. That is, without wishing to be bound by any particular theory or mechanism of action, macrophages may provide signals that modulate cardiac inflammation but changes in cardiomyocytes inflammation may not reciprocally affect macrophage signaling.

[0057] These observations have at least four implications. First, macrophage-induced inflammation is likely upstream of cardiomyocyte dysfunction in the pathogenesis of HFpEF. Second, by enhancing SPM production in cardiomyocytes, it is possible to have a cardiac specific improvement in inflammation without systemic immunomodulation that may have deleterious effects. Third, it is plausible that the cardiomyocyte NLRP3 inflammasome is involved in the pathogenesis of HFpEF. Finally, SPMs reduce oxidative modification of cMyBPC S-glutathionylation and improve HFpEF.

[0058] Human gene therapies have been approved for use in human subjects to treat diseases such as Duchenne muscular dystrophy, spinal muscular atrophy, and the COVID-19 virus. Cardiac-specific gene therapy has been undertaken in humans. Therefore, it is conceivable that 5-LOX could represent an achievable therapy directed at the mechanism of HFpEF. This disclosure provides proof-of-principle that producing a pro-resolving cardiac milieu can treat DM-associated HFpEF. FIG. 9 illustrates one exemplary scheme for improving diastolic function in DM hearts. Using a gene therapy approach, one can treat HFpEF while limiting the likelihood and / or extent of systemic immunomodulation.

[0059] Thus, in one aspect, this disclosure describes a viral vector that includes a cardiomyocyte-specific promoter and a polynucleotide that encodes an enzyme that mediates expression of one or more specialized pro-resolving mediators (SPMs).

[0060] While described herein in the context of exemplary embodiments in which the cardiomyocyte-specific promoter is an a-myosin heavy chain (a-MHC) promoter, the compositions and methods described herein can involve the use of any suitable promoter. Exemplary alternative promoters include, but are not limited to the promoter for myosin light chain 2 (MLC-2a and v), troponin T, cardiac troponin C (cTnC), beta MHC, ANF, POSTN, Tcf21, Nkx2.5, Mef2C-AHF, Isll, or SM22.

[0061] While described herein in the context of an exemplary embodiment in which the enzyme that mediates expression of one or more specialized pro-resolving mediators is arachidonate 5- lipoxygenase (5-LOX), the compositions and methods described herein can involve the use of any enzyme that mediates expression of one or more specialized pro-resolving mediators. Exemplary alternative enzymes include, but are not limited to, cyclooxygenase 2 (COX-2), arachidonate 5-lipooxygenase (ALOX-5), ALOX- 12 and ALOX- 15, or AL0X-15B.

[0062] While described herein in the context of an exemplary embodiment in which the viral vector is adeno-associated virus 9 (AAV9), the compositions and methods described herein can involve using any suitable viral vector. Exemplary alternative vital vectors include, but are not limited to, an adenovirus, other adeno-associated viruses (e.g., serotype 1, serotype 2, serotype 4, serotype 6, serotype 7, serotype 8, or AAVrh74), Herpes simplex virus type 1, a lenti virus, moloney murine leukemia virus (MoMLV), a retrovirus, coxsackievirus A21, coxsackievirus B3, dengue vims, feline immunodeficiency vims, human immunodeficiency vims, Kunjin vims, measles vims, poxvirus, or Semliki Forest vims.

[0063] In one or more embodiments, the nucleotide encoding the enzyme may be delivered without the use of a viral vector. In such embodiments, the polynucleotide (e.g., DNA) may be delivered using any method for delivering a polynucleotide to a target cell known to those of skill in the art.

[0064] In another aspect, the viral vector may be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the viral vector, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the viral vector without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0065] The viral vector may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.

[0066] Thus, the viral vector may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The viral vector may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including, but not limited to, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

[0067] A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the viral vector into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and / or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0068] The viral vector — or a pharmaceutical composition that includes the viral vector — may be administered to a subject having, or at risk of having, a condition treatable using the vital vector. “Treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and / or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is often initiated before a condition manifests in a subject.

[0069] The subject can be a human or a non-human animal such as, for example, a livestock animal, a laboratory animal, or a companion animal.

[0070] Treatment that is prophylactic — e.g., initiated before a subject manifests a symptom or clinical sign of the condition — is referred to herein as treatment of a subject that is “at risk” of having the condition. As used herein, the term “at risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” of a condition is a subject possessing one or more risk factors associated with the condition such as, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Accordingly, a composition can be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the composition is not administered, decreasing the severity of symptoms and / or clinical signs of the condition, and / or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and / or clinical signs of the condition compared to a subject to which the composition is not administered, and / or completely resolving the condition.

[0071] Thus, the method includes administering an effective amount of the composition to a subject having, or at risk of having, a particular condition. In this aspect, an “effective amount” is an amount effective to reduce, limit progression, ameliorate, or resolve, to any extent, a symptom or clinical sign related to the condition.

[0072] While described herein in the context of an exemplary embodiment in which the condition is heart failure with preserved ejection fraction (HFpEF), the compositions and methods described herein can involve use of the viral vector to treat any suitable condition. Exemplary alternative conditions treatable using viral vectors described herein include, but are not limited to, ventricular arrhythmias, atrial arrhythmias including atrial fibrillation, pericarditis, myocarditis, cardiomyopathy, heart failure, ischemic heart disease, coronary artery disease, rheumatic and / or nonrheumatic valvular heart disease.

[0073] The amount of viral vector administered can vary depending on various factors including, but not limited to, the specific the specific viral vector being administered, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute amount of the viral vector included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of the viral vector effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors. For example, certain viral vectors may be administered at the same dose and frequency for which the drug has received regulatory approval. In other cases, certain viral vectors may be administered at the same dose and frequency at which the drug is being evaluated in clinical or preclinical studies. One can alter the dosages and / or frequency as needed to achieve a desired level of clinical effect. Thus, one can use standard / known dosing regimens and / or customize dosing as needed.

[0074] In one or more embodiments, the method can include administering sufficient viral vector to provide a dose of, for example, from about 108vector genomes per kg (vgs / kg) to 1018vgs / kg to the subject, although in one or more embodiments the methods may be performed by administering the viral vector in a dose outside this range.

[0075] Thus, for example, the method can include administering to a human subject a minimum dose of at least 109vgs / kg, at least 2>< 109vgs / kg, at least 3x l09vgs / kg, at least 5x l09vgs / kg, at least 5.8*109vgs / kg, at least 1010vgs / kg, at least 5* IO10vgs / kg, at least 1011vgs / kg, at least 2x lOnvgs / kg, at least 5x lOnvgs / kg, at least 1012vgs / kg, at least 5x l012vgs / kg, at least 1013vgs / kg, at least 2x l013vgs / kg, at least 3 x l013vgs / kg, at least 3.5xl013vgs / kg, or at least 5xl013vgs / kg to the subject.

[0076] The method can include administering to a human subject a maximum dose of no more than 1018vgs / kg, no more than 5x 1017vgs / kg, no more than 3x l017vgs / kg, no more than 2x 1017vgs / kg, no more than 1.5x l017vgs / kg, no more than 1017vgs / kg, no more than 5x l016vgs / kg, no more than 2x 1016vgs / kg, no more than 1016vgs / kg, no more than 7.5x 1013vgs / kg, no more than 5x 1015vgs / kg, no more than 1015vgs / kg, no more than 5* 1014vgs / kg, or no more than 1014vgs / kg to the subject.

[0077] The method can include administering to a subject a dose that falls within a range having endpoints defined by any minimum dose set forth above and any maximum dose set forth above that is greater than the selected minimum dose. Thus, for example, the method can include administering to the subject a dose of at least 5.8x 109vgs / kg to no more than 7.5x 1015vgs / kg, at least 3.5xl013vgs / kg to no more than 1.5* 1017vgs / kg, at least 109vgs / kg to no more than 1018vgs / kg, etc., to the subject.

[0078] A single dose may be administered all at once, continuously for a prescribed period of time, or in multiple discrete administrations. When multiple administrations are used, the amount of each administration may be the same or different. For example, a dose of 5X1011vector genomes may be administered as a single administration of 5x lOnvector genomes, either simultaneously or continuously over a prescribed period of time (e.g., 24 hours), as two or more equal administrations (e.g., two administrations of 2.5* 1011vector genomes), or as two or more unequal administrations (e.g., a first administration of 4X1011vector genomes followed by a second administration of I xlO11vector genomes). When multiple administrations are used to deliver a single dose, the interval between administrations may be the same or different.

[0079] In one or more embodiments, the viral vector may be administered, for example, from a single dose to multiple doses per week, although in one or more embodiments the method can involve a course of treatment that includes administering doses of the viral vector at a frequency outside this range. When a course of treatment involves administering multiple doses within a certain period, the amount of each dose may be the same or different. For example, a course of treatment can include an initial loading dose, followed by a maintenance dose that is lower than the loading dose. Also, when multiple doses are used within a certain period, the interval between doses may be the same or be different.

[0080] Thus, the viral vector may be administered from a single once-off dose to about five times per week. Thus, the method can include administering the viral vector at a minimum frequency of a single dose, one dose per year, one does every six months, one dose every four months, one dose every three months, one dose every two months, or one dose every month. The method can include administering the viral vector at a maximum frequency of one dose per day, one dose every two days, one dose every three days, one dose per week, one dose every two weeks, one dose per month, or one dose every six months.

[0081] In one or more embodiments, the method can include administering the viral vector at a frequency that falls within a range having endpoints defined by any minimum frequency set forth above and any maximum frequency set forth above that is more frequent than the selected minimum frequency. Thus, for example, the viral vector may be administered to a subject at a frequency of from a once-off administration to once per week, from a once-off administration to once per month, from once per week to once every six months, from once per month to once every two weeks, etc.

[0082] The duration of therapy according to the methods described herein can be from a once-off administration to the lifetime of the subject. Thus, the duration of treatment can be a minimum of a once-off administration, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, or at least ten years. The duration of treatment can be a maximum of for the remining lifetime of the subject, no more than twenty years, no more than ten years, no more than five years, no more than two years, no more than one year, no more than six months, or no more than two months.

[0083] In one or more embodiments, the duration of treatment can fall within a range having endpoints defined by any minimum duration set forth above and any maximum duration set forth above that is greater than the selected minimum duration. Thus, the duration of treatment can be from a once-off administration to no more than ten years, from at least one month to the remaining lifetime of the subject, from at least one month to no more than five years, from at least six months to no more than two years, from at least six months to no more than one year, etc.

[0084] In another aspect, this disclosure provides a method of overexpressing 5-LOX in a subject. Generally, the method includes administering to the subject a pharmaceutical composition that includes any embodiment of viral vector encompassed by this disclosure that has a polynucleotide encoding arachidonate 5-lipoxygenase (5-LOX) operably linked to the promoter.

[0085] In another aspect, this disclosure provides a method of increasing expression of a proresolving mediator (SPM) in a subject. Generally, the method includes administering to the subject a pharmaceutical composition that includes any embodiment of viral vector encompassed by this disclosure that has a polynucleotide encoding an enzyme that mediates expression of the SPM. In one or more embodiments, the SPM is resolvin DI (RvDl).

[0086] In another aspect, this disclosure describes a method of reducing NLRP3 inflammasome expression in a subject. Generally, the method includes administering to the subject a pharmaceutical composition that includes any embodiment of viral vector encompassed by this disclosure effective to reduce NLRP3 inflammasome expression in the subject.

[0087] In another aspect, this disclosure describes a method of increasing inflammation resolution in a subject. Generally, the method includes administering to the subject a pharmaceutical composition that includes any embodiment of viral vector encompassed by this disclosure effective to increase resolution of inflammation in the subject. In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0088] As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0089] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0090] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0091] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0092] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0093] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0094] EXAMPLES

[0095] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0096] Sex as a biological variable

[0097] Only male diabetic mice were examined as female C57BL / 6J mice were less susceptible from high fat diet-induced (HFD-induced) diabetes mellitus (DM).

[0098] Animal experimental protocol

[0099] The HFD-induced diabetes mouse model has been previously described (Liu et al., 2023, J ACC Basic Transl Sci. 8(2): 174-85; Liu et al., 2019, JCI Insight. 4(1)). Briefly, male C57BL / 6J mice were fed with a HFD (60 kcal% fat, Research Diet, New Brunswick, NJ) for 20-21 weeks starting from six weeks of age and screened by fasting blood glucose level to confirm the development of DM using a glucometer (ACCU-CHEK, Roche Applied Science, Mannheim, Germany). Sex-matched and age-matched C57BL / 6J mice receiving normal chow (Harlan, Indianapolis, IN) served as the controls. At the age of 24 weeks, the DM mice were treated intraperitoneally with either resolvin DI (RvDl) at 200 ng / mouse (Cayman Chemical, #10012554) or USP normal saline daily for two weeks. To specifically upregulate arachidonate 5 -lipoxygenase (5-LOX) in cardiomyocytes, a cohort of DM mice received intravenous injection of an adeno-associated virus serotype 9 (AAV9) viral vectors (5* 10ugenome copies / mouse) at the age of 20 weeks under the direction of the a-myosin heavy chain (a-MHC) promoter. The age-matched control DM mice received the same copy number of AAV9-plain viral vectors.

[0100] Alternatively, male C57BL / 6J mice (The Jackson Laboratory, Strain #380050) were fed with a HFD (60 kcal% fat, Research Diet, New Brunswick, NJ) for 20-24 weeks starting from six-to-eight weeks of age and screened by fasting blood glucose level to confirm the development of DM using a glucometer (ACCU-CHEK, Roche Applied Science, Mannheim, Germany). Sex and age-matched C57BL / 6J mice (The Jackson Laboratory, Strain #000664) receiving normal chow (Envigo Teklad Diets, 2018 Teklad Global 18% Protein Rodent Diets) served as the controls. At the age of 29-30 weeks, the DM mice were treated intraperitoneally with RvDl at 100 ng / mouse (Cayman Chemical, #10012554, Ann Arbor, MI) daily for 2-3 weeks, as previously described (Markworth et al., JCI Insight. 2020). To specifically upregulate 5-LOX in cardiomyocytes, a cohort of DM mice received intravenous injection of adeno- associated viral vectors serotype 9 (AAV9) viral vectors (5xl0ngenome copies / mouse) at the age of 24-25 weeks under the direction of the a-myosin heavy chain (a-MHC) promoter and were followed for seven-to-eight weeks. The age-matched control DM mice received the same copy number of AAV9-plain viral vectors.

[0101] Echocardiographic evaluation of cardiac function

[0102] Echocardiography was performed using the VEVO 2100 (VisualSonics, Toronto, Canada) ultrasound system as previously described (Liu et al., 2023, JACC Basic Transl Sci. 8(2): 174-85; Liu et al., 2019, JCI Insight. 4(1)). Mice were anesthetized with 1-2% isoflurane in oxygen at 1 L / min with the body temperature maintained at 37 °C to 38 °C and the heart rate maintained above 400 bpm during the procedure. B-mode images along the left ventricular parasternal long axis and then M-mode images at the mid-papillary level were obtained to calculate ejection fraction and chamber size. To evaluate diastolic function, mitral inflow velocity (E) and longitudinal tissue velocity of the mitral anterior annulus (E ) were assessed in the subcostal 4-chamber view by pulsed-wave and tissue Doppler imaging to calculate E / E'. Measurements were averaged from five consecutive beats during expiration.

[0103] Protein immunoblots

[0104] Proteins from the left ventricles were isolated and separated on SDS-PAGE gels and transferred to 0.2 pm polyvinyl difluoride membranes. After incubation with 5% nonfat milk for one hour at room temperature, the membranes were incubated with the corresponding primary antibodies overnight at 4 °C (Cell Signaling Technology, Inc., Danvers, MA: anti-5-LOX, #3289, anti-CD68, #97778; anti-MCP-1, #2029; anti-NLRP3, #15101; anti-IL-10 #12242; anti- vinculin, #13901; Abeam: anti-GAPDH, #ab9484; anti-tubulin, #abl76560; Santa Cruz Biotechnology, Inc., Dallas, TX: anti-cMyBPC, #sc-137180; Virogen Biotechnology, Inc., Watertown, MA: anti-glutathione, #101-A), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, Inc., Hercules, CA) for one hour at room temperature. Protein bands were visualized by chemiluminescence detection, and the band optical density was analyzed with IMAGE LAB 6.0.0 software (Bio-Rad Laboratories, Inc., Hercules, CA).

[0105] ELISA assay for IL- 10 and RvDl measurements

[0106] Approximately 25 mg of mouse left ventricle was homogenized in tissue protein extraction reagent (Thermo Fisher Scientific, Inc., Waltham, MA) containing a protease / phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc., Waltham, MA). The supernatant was collected, and the IL-10, LTA4, and RvDl levels were determined by commercially available ELISA kits (Mouse IL-10 / IL-1F2 Quantikine ELISA Kit, #MLB00C; Cayman Chemical, Resolvin DI ELISA KIT, #500380, R&D Systems, Inc., Minneapolis, MN; LTA4 ELISA KIT, Abbexa, Ltd., Houston, TX) according to the manufacturer’s protocol.

[0107] Statistics

[0108] Continuous data were presented as mean ± standard error of the mean (SEM). For the dot plots, the lines indicated the mean values, and the error bars indicated SEM. Data were analyzed using a 2-tailed Student’s t test for comparisons between two groups. All statistical analyses were performed with PRISM 5.0 (GraphPad Software, San Diego, CA). A P value < 0.05 was considered statistically significant.

[0109] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0110] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0111] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0112] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A viral vector comprising: a cardiomyocyte-specific promoter; and a polynucleotide operably linked to the cardiomyocyte-specific promoter, the polynucleotide encoding an enzyme that mediates expression of one or more specialized proresolving mediators.

2. The viral vector of claim 1, wherein the cardiomyocyte-specific promoter comprises an a- myosin heavy chain (u-MHC) promoter or a troponin T promoter.

3. The viral vector of claim 1 or claim 2, wherein the viral vector further comprises an adeno- associated virus serotype 1 (AAV1), an adeno-associated virus serotype 2 (AAV2), or an adeno- associated virus serotype 9 (AAV9).

4. The viral vector of any preceding claims, wherein the enzyme that mediates expression of one or more specialized pro-resolving mediators is arachidonate 5 -lipoxygenase (5-LOX).

5. A pharmaceutical composition comprising: a viral vector comprising: a cardiomyocyte-specific promoter; and a polynucleotide operably linked to the cardiomyocyte-specific promoter, the polynucleotide encoding an enzyme that mediates expression of one or more specialized pro-resolving mediators; and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the cardiomyocyte-specific promoter comprises an a-myosin heavy chain (a-MHC) promoter or a troponin T promoter.

7. The pharmaceutical composition of claim 5 or claim 6, wherein the viral vector further comprises an adeno-associated virus serotype 1 (AAV1), an adeno-associated virus serotype 2 (AAV2), or adeno-associated virus serotype 9 (AAV9).

8. The pharmaceutical composition of any one of claims 5-7, wherein the enzyme that mediates expression of one or more specialized pro-resolving mediators is arachidonate 5 -lipoxygenase (5-LOX).

9. A method of overexpressing 5-LOX in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 8.

10. A method of increasing expression of a pro-resolving mediator (SPM) in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

11. The method of claim 10, wherein the SPM is resolvin DI (RvDl).

12. A method of reducing NLRP3 inflammasome expression in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

13. A method of reducing inflammation in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

14. A method of increasing inflammation resolution in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

15. A method of treating heart failure with preserved ejection fraction (HFpEF) in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

16. A method of treating diastolic dysfunction in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

17. A method of reducing oxidative modification of cMyBPC S-glutathionylation in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 5-8.

18. The method of any one of claims 15-17, wherein the subject has diabetes mellitus.

19. The method of any one of claims 15-17, wherein the subject has a high fat diet.

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