Protein kinase A inhibitory peptide and its uses

Abstract

This relates to engineered PKA repressive peptides, dimers or polymers formed by the binding of said monomers. It further relates to the use of said PKA repressive peptides.

Description

Protein kinase A inhibitory peptide and use thereof Technical Field

[0001] The present application relates to the field of cardiovascular disease treatment, and in particular to an inhibitory peptide capable of inhibiting protein kinase A, a nucleic acid construct encoding the same, and uses thereof. Background Art

[0002] Damage to myocardial cells (such as those caused by ischemia, hypertension, metabolic diseases, aortic valve regurgitation or genetic diseases) feedback activates multiple neuroendocrine systems to maintain cardiac output and meet the body's blood perfusion needs. The activation of the sympathetic-adrenal medullary system is the core mechanism. Although the activation of this system can increase heart rate, strengthen myocardial contractility and accelerate cardiac relaxation, thereby achieving the goal of maintaining cardiac output, the continuous and long-term activation of this system causes adverse changes in the morphology, structure and function of the myocardium, namely adverse remodeling, which ultimately leads to heart failure and malignant arrhythmias. It is generally believed that catecholamines released by the sympathetic-adrenal medullary system act on the beta-receptors of the heart, activating downstream signals cAMP / PKA and Ca 2+ / CaM / CaMKII is toxic to the heart and is one of the main causes of heart disease.

[0003] Our research shows that the patented improved protein kinase A inhibitory peptide (PKAi) is the PKA inhibitory domain of the α-type cAMP-dependent protein kinase inhibitory peptide (PKIα). It can prevent the activation of CaMKII by preventing the activation of cytoplasmic PKA and the increase of calcium when myocardial cells are damaged, while activating the protective cAMP / EPAC signal, protecting myocardial cells, inhibiting cardiac remodeling, and thus improving the structure and function of the damaged myocardium.

[0004] Summary of the Invention

[0005] The present invention utilizes amino acids 1-25 of the α-type cAMP-dependent protein kinase inhibitory peptide (PKIα) as a base sequence for further engineering, enhancing the activity of this protein kinase A inhibitory peptide. Furthermore, to ensure its stability, peptides that do not affect its activity are added before and after PKAi, or two PKAi monomers are linked using a linker sequence to form a PKAi dimer, achieving greater stability compared to traditional monomers. Furthermore, to maximize the efficacy of the protein kinase A inhibitory peptide while minimizing its toxicity, a core sequence is added to the N-terminus or C-terminus of the protein kinase A inhibitory peptide, based on its stable monomer or dimer. Specific expression of this peptide in cardiomyocytes can alleviate cardiomyocyte damage, inhibit adverse remodeling, and restore functional function, while reducing cardiomyocyte toxicity.

[0006] In one embodiment of the present invention, the monomers in the PKAi dimer are connected by covalent bonds or linkers, and the linkers can be cleavable linkers and non-cleavable linkers, wherein the cleavable linker can be RKRRKR (SEQ ID NO: 18), GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 6), GFLG (SEQ ID NO: 19), etc.; the non-cleavable linker is also called an "internal linker", for example, GGGSGGGSGGGS (SEQ ID NO: 5); (GGGGS)3 (SEQ ID NO: 12); (G)8 (SEQ ID NO: 13); (G)6 (SEQ ID NO: 14); (EAAAAK)3 (SEQ ID NO: 15); PAPAP (SEQ ID NO: 16); AEAAAKEAAAKA (SEQ ID NO: 17); ((G)m(S)n)p linker, wherein m, n or p is any positive integer selected from 1-20; preferably, the linker is a cleavable linker.

[0007] The "internal linker" herein refers to a chemical structure fragment that is connected to one PKAi monomer at one end and to another PKAi monomer at the other end. In the present invention, the "internal linker" between PKAi monomers can be an enzyme-stable linker.

[0008] In one embodiment of the present invention, PKAi can form a multimer, wherein one form of the multimer is a plurality of PKAi monomers connected by an "internal linker". A second form of the multimer is a plurality of PKAi monomers connected by a "cleavable linker" (a "cleavable linker" is a polypeptide covalently linked to a PKAi monomer, which can be specifically and efficiently cleaved by an intracellular protease). A third form of the multimer is a plurality of "PKAi dimers" connected by a "cleavable linker". A fourth form of the multimer is a free combination of PKAi monomers and PKAi dimers, and its linker can be an "internal linker" or a "cleavable linker".

[0009] In one embodiment of the present invention, a "cleavable linker" specifically refers to a linker that can be effectively cut by intracellular proteases such as furin. This peptide connection is believed to be very stable in the plasma circulation because proteases are generally inactive outside the cell due to extracellular serum protease inhibitors. In view of the high plasma stability and good intracellular cleavage selectivity and effectiveness. The "cleavable linker" comprises a peptide with an intracellular enzyme cleavage site, specifically, for example, GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 6), GFLG (SEQ ID NO: 19), KRRKR (SEQ ID NO: 20), etc., which are cleaved by intracytoplasmic proteases such as furin.

[0010] In one embodiment of the present invention, a nuclear export sequence (NES) is added to the N-terminus or C-terminus of the PKAi dimer sequence to prevent potential harmful effects in the nucleus. A nuclear export sequence (also known as a nuclear export signal, NES) is an amino acid sequence on a protein containing four hydrophobic groups that is responsible for transporting proteins from the nucleus to the cytoplasm through the nuclear pore. The NES has the opposite function to the nuclear localization signal (NLS), a domain on a protein responsible for transporting large proteins into the nucleus. The NES fragment is recognized and bound by karyopherins. Once bound, the resulting Ran-karyopherin-target protein complex crosses the nuclear membrane through the nuclear pore. In the present invention, the nuclear export sequence can be, for example, sequences that mark polypeptide nuclear export, such as LQKKLEELEL (SEQ ID NO: 8), LECLNEQRLQGL (SEQ ID NO: 21), LRAARLRHQELFR ​​(SEQ ID NO: 22), or LPPLERLTL (SEQ ID NO: 23). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. Artificially constructed cardiomyocyte-specific PKAi monomer expression vector.

[0012] Figure 2. Artificially constructed cardiomyocyte-specific PKAi dimer or multimer expression vector.

[0013] Figure 3. Effects of AAV9.hTNT.PKAi-dimer-NES on the prevention and treatment of heart failure after myocardial infarction. C57 / bl6 mice underwent myocardial infarction surgery and were injected intravenously with 5E10 vg / mouse one day later. A. Ejection fraction in the treated and untreated groups. B. Percentage of left ventricular systolic thickening in the treated and untreated groups. *: p < 0.05; **: p < 0.001, comparison between the "MI + AAV9.PKAi dimer" and "MI" groups.

[0014] Figure 4. AAV9.hTNT.PKAi-dimer-NES prevents adverse cardiac remodeling after myocardial infarction. C57 / bl6 mice were injected with 5E10 vg / mouse via the tail vein one day after myocardial infarction. Changes in cardiac output (A), end-diastolic interventricular septal thickness (B), end-diastolic left ventricular posterior wall thickness (C), and end-diastolic left ventricular internal diameter (D) in treated and untreated groups over time after myocardial infarction. *: p < 0.05; **: p < 0.001, comparison between the "MI + AAV9.PKAi-dimer" and "MI" groups.

[0015] Figure 5. AAV9.hTNT.PKAi-GFP prevents and treats pressure-overload-induced left ventricular hypertrophy and heart failure. A. Mouse pressure-overload model and treatment regimen. One day after transaortic constriction (TAC) or sham surgery, mice were injected with saline (PBS) or AAV (2E11 vg / animal) via the tail vein. The inset below shows FLAG staining after AAV infection (with a 6x FLAG sequence inserted after GFP). B. Percentage of mice surviving after surgery. C. Heart rate at the time of echocardiography. D. Corrected left ventricular mass measured by echocardiography at different time points after surgery. E. End-diastolic left ventricular internal diameter at different time points after surgery. F. Changes in ejection fraction at different time points after surgery. G. Heart-to-body weight ratio after animal sacrifice. H. Lung-to-body weight ratio after animal sacrifice. Numbers in the figure indicate p values ​​for the aortic constriction / AAV treatment group compared with the aortic constriction / PBS control group.

[0016] Figure 6. AAV1.hTNT.PKAi-GFP prevents right ventricular hypertrophy and heart failure after hypoxia-induced pulmonary hypertension. Mice were injected with AAV (5E10 vg / animal) 2 hours after hypoxia-induced pulmonary hypertension. Right ventricular structure and function were assessed weekly by echocardiography. A. Changes in right ventricular systolic area over treatment time. B. Changes in right ventricular free wall thickness over treatment time. C. Changes in tricuspid annular systolic excursion over time. *or # : Statistical difference between curves p<0.05 or p<0.01.

[0017] Figure 7. AAV1.hTNT.PKAi-GFP prevents right ventricular myocardial fibrosis and hypertrophy after hypoxia-induced pulmonary hypertension. After treatment with pulmonary hypertension, hearts were fixed, sectioned, and stained with hematoxylin and eosin, Masson's stained, or wheat germ agglutinin (WGA). A. Transverse section of the heart, hematoxylin and eosin staining. Hypoxia induces right ventricular free wall thickening (left two images), an effect that is inhibited by AAV1-hTNT-PKAi-GFP (right two images). B. Magnified image of the right ventricular free wall, Masson's stained, demonstrates that hypoxia induces myocardial fibrosis in the AAV1-hTNT-GFP control group, but AAV1-hTNT-PKAi-GFP prevents this fibrosis. C. WGA staining of the myocardium. Hypoxia induces an increase in myocardial cross-sectional area in the AAV1-hTNT-GFP control group, but AAV1-hTNT-PKAi-GFP prevents this change. D. Ratio of right ventricular free wall mass to left ventricular total mass (left ventricular free wall mass + interventricular septal mass). Hypoxia induced an increase in this ratio in the control-treated group, indicating right ventricular hypertrophy; however, AAV1.PKAi-GFP attenuated this hypertrophic effect. E. Hypoxia stimulated an increase in cardiomyocyte cross-sectional area in the control-treated group (AAV1.GFP), but AAV1.PKAi-GFP prevented this increase. Specific implementation plan

[0018] The purpose of the present invention is to provide an expression frame for stably and efficiently expressing protein kinase inhibitory peptides for treating heart diseases in view of the deficiencies in the existing technology.

[0019] The second object of the present invention is to provide a vector and a vector system containing the expression cassette.

[0020] The third object of the present invention is to provide a recombinant adeno-associated virus expressing a protein kinase inhibitory peptide and a composition thereof.

[0021] The fourth object of the present invention is to provide the use of the gene expression cassette, vector, and recombinant adeno-associated virus in treating heart diseases.

[0022] The purpose of the present invention can be achieved through the following technical solutions:

[0023] A. The protein kinase A inhibitory peptide of the present invention (hereinafter referred to as PKAi) comprises the protein kinase A inhibitory domain of PKIα, such as the amino acid sequence of amino acids 1 to 25 shown in SEQ ID NO: 1, which is derived from the human PKAi protein sequence (SEQ ID NO: 11) and is further modified.

[0024] In some embodiments, a linker sequence (e.g., SEQ ID NO: 5 or SEQ ID NO: 6) may be added to the modified PKAi, and a second PKAi fragment may be attached to the linker to form a dimer (e.g., SEQ ID NO: 7). The PKAi sequence may be any of two variants based on SEQ ID NO: 1. The nucleotide sequence encoding the second PKAi fragment does not contain a start codon but contains a stop codon. In some embodiments, the present invention can further add a linker sequence (e.g., SEQ ID NO: 5 or SEQ ID NO: 6) based on the modified PKAi, and connect a second PKAi, a third PKAi, a fourth PKAi, a fifth PKAi, a sixth PKAi, a seventh PKAi, or an eighth PKAi, etc. after the linker to form a dimer (e.g., SEQ ID NO: 7), a trimer, a tetramer, a pentamer, a hexamer, a heptamer, or an octamer, or more multimers, wherein the PKAi sequence can be any variant based on SEQ ID NO: 1 (sequence 1).

[0025] As a preferred embodiment of the present invention, we mutated the tyrosine at position 8 of the PKAi sequence to phenylalanine (SEQ ID NO: 2). Studies have shown that this mutation improves the activity and stability of PKAi.

[0026] As a preferred embodiment of the present invention, we mutated the serine at position 14 of the PKAi sequence to cysteine ​​(SEQ ID NO: 3). Studies have shown that this mutation improves the activity and stability of PKAi.

[0027] As a preferred embodiment of the present invention, we mutated the tyrosine at position 8 of the PKAi sequence to phenylalanine and the serine at position 14 to cysteine ​​(SEQ ID NO: 4), which further improved the activity and stability of PKAi.

[0028] B. To better express protein kinase inhibitory peptides in the heart, we used human troponin T or human brain natriuretic peptide promoters when constructing expression cassettes. Studies have shown that these promoters can specifically activate the expression of protein kinase inhibitory peptides in cardiomyocytes or at specific disease stages.

[0029] C. As a preferred embodiment of the present invention, we provide a solution to add three consecutive nuclear export sequences (NES, such as SEQ ID NO: 8) after the PKAi dimer sequence. Studies have found that this solution improves the function of protein kinase A inhibitors and reduces their potential toxic effects.

[0030] D, A WPRE sequence is added after the protein kinase A inhibitory peptide dimer to maintain sequence stability.

[0031] E. As a vector, the vector used for the present expression cassette includes but is not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any other AAV vector now known or later discovered.

[0032] This patent uses a cardiac-specific promoter to improve the specificity, efficiency and disease responsiveness of gene expression in cardiomyocytes. The gene used in this patent is an upstream regulatory molecule that can regulate multiple cardiac function regulatory genes including SERCA2a, L-type calcium channels, CaMKII, etc. by inhibiting PKA. Therefore, a very low viral dose is required to achieve a higher regulatory effect.

[0033] Currently synthesized PKAi analogs include:

[0034] PKAi-(6-22)-amide(Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile -NH2)(Anal.Bioanal.Chem.381,647–655.doi:10.1007 / s00216-005-3070-2),PKAi-(14-24)-amide

[0035] (Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-NH2)(Apoptosis 11,1263–1273.doi:10.1007 / s10495-006-7702-6) and

[0036] PKAi-(5-24)-amide (Thr-Thr-Tyr-Ala-Asp-PheIle-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-NH2) (Gen. Comp. Endocrinol. 181, 88–97. doi: 10.1016 / j.ygcen.2012.10.016). All of them have poor intracellular stability and short duration of effect. The inhibitory efficiency against PKA is PKAi-(6-22) > PKAi-(5-24) > PKAi-(14-24) from high to low.

[0037] Currently, the protein kinase A inhibitory peptide sequences available on the market include three lengths: PKAi-(6-22), PKAi-(5-24) and PKAi-(14-24), all of which are artificially synthesized.

[0038] The α-type PKI (PKIα) consists of 75 amino acids, of which 1-25 amino acids constitute the PKA inhibitory domain (PKAi) of PKIα.

[0039] Currently, recombinant adeno-associated viruses are prepared and purified by co-expressing accessory elements, capsid proteins, and genomic DNA containing a target gene expression cassette in a human embryonic kidney (HEK293) cell line. Viral particles are then continuously packaged within the cells and subsequently lysed for viral extraction. In the laboratory, viable viral particles can be purified using density gradients using iodixanol; however, large-scale production utilizes chromatography column-based separation and purification methods.

[0040] Example

[0041] 1. Materials and Methods

[0042] 1.1 Instrument Information

[0043] 1.2 Reagent Information

[0044] 1.3. Specific experimental methods

[0045] 1.3.1 Plasmid construction

[0046] The optimized AAV main plasmid, auxiliary plasmid, pAAV.hTNT.PKAi-GFP.SV40\pAAV.hTNT.GFP.SV40 or pAAV.hTNT.PKAi dimer.SV40, pAAV2 / 1 or pAAV2 / 9n and pAdDeltaF6 were all artificially synthesized in full length.

[0047] 1.3.2 Virus packaging and purification

[0048] AAV was prepared according to the experimental method of Grieger et al. (Nat Protoc. 2006; 1(3): 1412-28).

[0049] 1.3.3 AAV virus gene extraction and titer detection:

[0050] Add 100 μl of DNase lysis solution (50 U DNase I + 1 mL DNase lysis solution) to 2 μl of virus solution.

[0051] Mix thoroughly for 2 seconds, being careful not to damage the viral coat. Incubate in a 37°C water bath for 1 hour.

[0052] Add 5 μL of EDTA, mix well, and place in a metal bath at 70°C for 10 min.

[0053] Add 120 μL of protease lysis solution (1.818 U proteinase K + 500 μL proteinase K solution), mix well, centrifuge for 3 seconds, and treat in a 55°C metal bath for 2 hours.

[0054] The metal bath temperature was adjusted to 95°C for 10 min to inactivate the protease and the cells were placed on ice.

[0055] The obtained sample was diluted, and 3 μL was taken and diluted 300 times for titer detection.

[0056] The extracted DNA was quantified by RT-PCR and standard plasmids to determine the AAV virus titer.

[0057] 1.3.4 Echocardiography

[0058] Myocardial Infarction Model: AAV9, hTNT, and PKAi-dimer-NES were injected into each mouse at a dose of 5E10 vg on the day following myocardial infarction surgery. Echocardiography was performed weekly postoperatively to assess left ventricular function.

[0059] Pulmonary hypertension model: In the hypoxia model, virus injection was performed two weeks after the model was established. The injected virus was AAV1.hTNT-PKAi-GFP, and the injection volume was 5E10 vg per mouse. Echocardiography was performed every two weeks from the establishment of the model.

[0060] Example 1: Human protein kinase inhibitory peptide gene sequence optimization and vector construction

[0061] The TNT promoter sequence is from NCBI Reference Sequence: NG_007556.1 (4499bp-5042bp, as shown in SEQ ID NO: 9). The coding sequence of the 1-25 amino acids of the human protein kinase inhibitory peptide sequence was optimized and synthesized into the pAAV-hTNT-PKAi-dimer-3NES vector (Figure 2). In addition, PKAi-dimer-NES units can be used as units to prepare PKAi polymers (such as 4-mers, 6-mers, etc.), and the units are connected by short peptide sequences that can be cleaved by proteases in the cytoplasm. Alternatively, PKAi-dimer-NES units and PKAi-monomer units can be mixed and combined to form a polymer (Figure 2).

[0062] Example 2: Packaging and purification of AAV9.hTNT-PKAi-dimer-NES virus

[0063] Refer to the viral packaging, purification, and titer detection methods reported by Grieger et al. (Nat Protoc. 2006; 1(3): 1412-28) and Rosemary C. Challis (dx.doi.org / 10.17504 / protocols.io.84ahyse). The Rep-Cap expression plasmid (pAAV2 / 9), helper plasmid (pAdΔF6), and protein kinase inhibitor peptide expression plasmid (pAAV-hTNT-PKAi-dimer-NES) were co-transfected into HEK293T cells using PEI for viral element expression and packaging. After 96 hours of culture, the cell culture medium and cells were harvested, the cell culture medium was precipitated with PEG8000, and the cell lysate was mixed and centrifuged to obtain a crude virus solution. Virus was purified using iodixanol density gradient centrifugation and titer was determined using qPCR.

[0064] Example 3: Myocardial infarction model establishment and treatment

[0065] We established a mouse myocardial infarction (MI) model and injected AAV9.PKAi dimer (SEQ ID NO: 7)-NES virus (5E10 vg / mouse) after surgery. Echocardiography was then performed weekly. At the fourth week of observation, we found that the ejection fraction, ventricular septal systolic thickness / diastolic thickness ratio (i.e., the degree of ventricular septal systolic thickening), and cardiac output of the treated mice were significantly improved (see Figures 3A and B, Figure 4A). When evaluating the left ventricular structure, echocardiography showed that the end-diastolic thickness of the ventricular septum and left ventricular posterior wall did not change significantly in the treated group, while the ventricular thickness in the non-treated group was thinner (Figures 4B and C). Myocardial infarction can lead to an increase in left ventricular internal diameter, and we found that the treatment group improved the expansion of left ventricular internal diameter (see Figure 4D). These results indicate that protein kinase inhibitory peptide dimers can improve cardiac dysfunction and remodeling caused by myocardial infarction.

[0066] Example 4: Construction of human protein kinase inhibitory peptide fused with green fluorescent protein (GFP) expression vector

[0067] The hTNT promoter sequence was derived from NCBI Reference Sequence: NG_007556.1 (4499 bp-5042 bp). The 1-25 amino acids of the human protein kinase inhibitory peptide sequence were synthesized and then connected to the full-length GFP sequence via a linker sequence. The pAAV-hTNT-PKAi-GFP vector was constructed after enzyme digestion and ligation (Figure 1A). At the same time, a control viral GFP expression vector pAAV-hTNT-GFP was constructed.

[0068] Example 5: Packaging and purification of AAV1-hTNT-PKAi-GFP or AAV9-hTNT-PKAi-GFP viruses

[0069] HEK293T cells were co-transfected with a Rep-Cap expression plasmid (pAAV2 / 1 or pAAV2 / 9n), a helper plasmid (pAdΔF6), and a protein kinase inhibitory peptide expression plasmid (pAAV-hTNT-PKAi-GFP) via PEI for viral expression and packaging. After 96 hours of culture, the cell culture medium and cells were harvested, precipitated with PEG8000, and mixed with the cell lysate and centrifuged to obtain a crude viral suspension. Virus was purified using iodixanol density gradient centrifugation and titered by qPCR.

[0070] Example 6: Establishment and treatment of aortic stenosis (TAC)-induced left ventricular hypertrophy and heart failure model

[0071] We surgically established a model of ascending aortic stenosis to increase left ventricular afterload, induce left ventricular hypertrophy, and heart failure. Gene therapy with AAV9-hTNT-PKAi (SEQ ID NO: 1)-GFP reversed the hypertrophy and prevented heart failure. A median sternotomy was performed to visualize the transverse aortic arch. A 7-0 silk ligature was placed around the aorta between the right brachiocephalic and left common carotid arteries using a 26-gauge needle. The needle was then removed to constrict the aorta and increase afterload. Sham surgery was performed on animals as a control for TAC. Following surgery, AAV9.hTNT.PKAi-GFP was injected via the tail vein at a dose of 2E11 vg / mouse. Animals were studied for 6 weeks. Survival was assessed daily. Echocardiography (ECHO) was performed weekly on anesthetized mice (1.5% isoflurane) using a VisualSonics Vevo 2100 instrument (FUJIFILM Visualsonic, Toronto, Canada) to assess cardiac function and morphology. ECHO baseline heart rate was maintained between 400 and 450 beats per minute (Figure 5C). Immunohistochemical staining demonstrated that over 95% of cardiomyocytes were infected and expressed PKAi-GFP (Figure 5A). AAV9.hTNT.PKAi-GFP-treated mice had significantly improved survival compared to untreated mice (Figure 5B). While heart rates were largely consistent between control groups (Figure 5C), gene therapy significantly reduced or prevented TAC-induced increases in left ventricular mass (Figure 5D), left ventricular end-diastolic diameter dilation (Figure 5E), and decreased ejection fraction (i.e., heart failure) (Figure 5F), as well as the ratio of heart weight to body weight (Figure 5G) and lung weight to body weight (Figure 5H). Numbers in the figures indicate p values ​​for the aortic stenosis / AAV-treated group compared to the aortic stenosis / PBS control group.

[0072] Example 7: Establishment and treatment of pulmonary arterial hypertension (PAH) model induced by SU5416 combined with hypoxia

[0073] We established a SU5416- and hypoxia-induced pulmonary artery hypertension (PAH) model and administered gene therapy using either AAV1-hTNT-PKAi (SEQ ID NO: 2)-GFP or AAV1.hTNT-GFP (AAV dose: 5E10 vg / mouse). Echocardiographic assessments at weeks 0, 2, 4, 6, and 8 revealed that AAV1.hTNT-PKAi-GFP significantly improved PAH-induced right ventricular dysfunction (defined as a decrease in the percentage change in right ventricular area) (Figure 6A). Furthermore, PAH-induced right ventricular free wall thickening was significantly reduced (Figure 6B). Echocardiographic assessments at week 8 revealed a significant increase in right ventricular tricuspid valve systolic excursion following PKAi-GFP treatment (Figure 6C).

[0074] After 8 weeks of PKAi treatment, samples were taken from the mice and the hearts were weighed. The right ventricle, left ventricle, and ventricular septum were then weighed separately. We found that the proportion of the right ventricle to the total weight of the left ventricle and ventricular septum was significantly increased in the pulmonary hypertension group, but significantly decreased after PKAi-GFP treatment (Figure 7D). H&E staining of fixed cardiac sections also showed consistent results: after pulmonary hypertension, right ventricular wall thickness increased, which was ameliorated after PKAi-GFP gene therapy (Figure 7A).

[0075] We sectioned and Massons' stained heart tissue and found that under normoxic conditions, no significant fibrosis was observed in either virus-injected group. Pulmonary hypertension led to increased right ventricular fibrosis, whereas PKAi gene therapy reduced the area of ​​right ventricular fibrosis (Figure 7B).

[0076] Wheat germ agglutinin (WGA) specifically binds to glycoproteins on the surface of cardiomyocyte membranes, staining them. We stained cardiomyocyte sections with WGA (Figure 7C) and counted the area of ​​cells containing DAPI. At least four fields of view were counted for each tissue, with a total of 100 cells counted and the average calculated. The statistical results showed that after pulmonary hypertension, right ventricular cardiomyocytes significantly hypertrophied, and this hypertrophy was significantly ameliorated after AAV1.hTNT-PKAi-GFP treatment (Figure 7E).

[0077] Sequence Listing

[0078] Note: The polynucleotide sequences in the above sequence listings may represent DNA sequences or RNA sequences. When representing RNA sequences, t represents uridine.

Claims

1. A protein kinase A inhibitory peptide (PKAi) monomer, comprising a protein kinase A inhibitory domain shown in formula (I): X0TDVETTX1ADX2IAX3GRTGRRNAIHD (I), Among them, X0 is absent or is methionine (M), and X1, X2 and X3 are each independently any amino acid.

2. The protein kinase A inhibitory peptide (PKAi) monomer according to claim 2, wherein: X1 is threonine (T), serine (S), tyrosine (Y), tryptophan (W) or phenylalanine (F), alanine (A), X2 is tyrosine (Y), phenylalanine (F), alanine (A), valine (V), leucine (L) or isoleucine (I), and X3 is serine (S), threonine (T) or cysteine (C), preferably the engineered PKI monomer comprises one of SEQ ID NO: 1-4.

3. The protein kinase A inhibitory peptide (PKAi) monomer according to claim 1 or 2, which further comprises at least one, two or three nuclear export sequences (NES) at the N-terminus and / or C-terminus, preferably the NES comprises the amino acid sequence shown in SEQ ID NO:

8.

4. A multimer formed by the protein kinase A inhibitory peptide (PKAi) monomer according to any one of claims 1-3, the multimer being a dimer, trimer, tetramer, pentamer, hexamer or higher multimer of the monomer.

5. The multimer according to claim 4, wherein the protein kinase A inhibitory peptide (PKAi) monomers are linked by covalent bonds or linkers.

6. The multimer according to claim 5, wherein the linker is selected from: 2A peptide and ((G)m(S)n)p, where m, n or p are independently any positive integer between 1 and 20.

7. The multimer according to claim 5, wherein the linker comprises a peptide having an intracellular enzyme-cleavable site.

8. The multimer according to claim 7, wherein the peptide having an intracellular enzyme-cleavable site comprises any one or more of SEQ ID NO: 6 and 19-20.

9. The multimer according to any one of claims 3-7, which comprises the amino acid sequence shown in any one of SEQ ID NO: 1-4.

10. The multimer according to any one of claims 4-10, which further comprises at least one, two or three or more nuclear export sequences (NES) at the N-terminus or C-terminus, preferably the NES comprises the amino acid sequence shown in SEQ ID NO:

8.

11. A nucleic acid molecule, which comprises a coding nucleic acid sequence of the protein kinase A inhibitory peptide (PKAi) monomer according to any one of claims 1-3 or the multimer according to any one of claims 4-10.

12. The nucleic acid molecule according to claim 11, further comprising a cardiac-specific or non-specific promoter nucleic acid sequence at the 5' end of the coding nucleic acid sequence. Preferably, the promoter is selected from one or more of the following cardiac-specific promoters: troponin T (TNT) promoter, brain natriuretic peptide (BNP) promoter, myosin light chain 2v (MLC2v) promoter, α-myosin heavy chain promoter.

13. The nucleic acid molecule according to claim 12, wherein the promoter comprises a polynucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO:

10.

14. The nucleic acid molecule according to any one of claims 11-13, may further comprise a coding sequence of WPRE at the 3' end of the coding nucleic acid sequence.

15. A genetic engineering vector, comprising the nucleic acid molecule according to any one of claims 11-14 or its complementary sequence. Preferably, the vector is a plasmid or a viral vector.

16. The genetic engineering vector according to claim 15, wherein the viral vector is a lentivirus or an adeno-associated virus (AAV) vector, such as AAV1, AAV8 or AAV9.

17. A genetic engineering cell, comprising the nucleic acid molecule according to any one of claims 11-14 or the genetic engineering vector according to claim 15 or 16.

18. A pharmaceutical composition, comprising the protein kinase A inhibitory peptide (PKAi) monomer according to any one of claims 1-3 or the multimer according to any one of claims 4-10, and a pharmaceutically acceptable carrier or excipient.

19. A method for preventing or treating myocardial injury diseases, comprising administering to a subject an effective amount of the pharmaceutical composition according to claim 18. Preferably, the myocardial injury disease is a myocardial injury disease caused by a cause selected from the following: ischemia, arterial hypertension, metabolic disorders, bacterial or viral infections, genetics.

20. The method of claim 19, wherein the myocardial injury disease is selected from one or more of the following: heart failure, myocardial infarction, myocardial hypertrophy, hypertension, pulmonary hypertension, sepsis and diabetes.