Targeting the renin-angiotensin system for treatment of environment-and pathogen-induced lung injury

Abstract

The present disclosure relates to the design and method for targeting the renin-angiotensin system (RAS) for therapeutics of lung diseases, particularly environment- and pathogen-induced lung injury. Provided herein are design, methodology, compositions, and the like for such for restoring the regulatory balance the RAS system for the management of lung diseases.

Claims

1. A method for treating environment- or pathogen-induced lung injury in a subject in need thereof by increasing Angiotensin-converting enzyme 2 (ACE2) expression, comprising injecting the subject with a recombinant ACE2 protein or segment of biologically active ACE2 protein.

2. The method of claim 1, wherein the subject is human.

3. The method of claim 2, wherein the subject is in need of treatment for SARS-coronavirus (SARS-Cov), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

4. The method of claim 1, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

5. A method for treating environment- or pathogen-induced lung injury in a subject in need thereof by compensating functional Angiotensin-converting enzyme 2 (ACE2) deficiency, comprising delivering to the subject one or more therapeutic vectors expressing ACE2 in lung tissue of the subject.

6. The method of claim 5, wherein the therapeutic vector is chosen from integrating or non-integrating lenti- and retro-viral vectors, AAV (Adeno-associated virus) vectors, and other gene therapy vectors that can be deliver genes into lungs.

7. The method of claim 6, wherein the subject is human.

8. The method of claim 7, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

9. The method of claim 6, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

10. The method of claim 5, wherein the subject is human.

11. The method of claim 10, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

12. The method of claim 5, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

13. A method for treating environment- or pathogen-induced lung injury in a subject in need thereof, comprising administering to the subject a composition comprising one or more active ingredients chosen from a therapeutic Ang-(1-7) heptapeptide, an ACE inhibitor, and a type I angiotensin II receptor blocker.

14. The method of claim 13, wherein the ACE inhibitor is lisinopril and wherein the type I angiotensin II receptor blocker is losartan.

15. The method of claim 14, wherein the subject is human.

16. The method of claim 15, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

17. The method of claim 14, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

18. The method of claim 13, wherein the subject is human.

19. The method of claim 18, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov).

20. The method of claim 20, wherein the subject is human, wherein the subject is in need of treatment for SARS-coronavirus (SARS-CoV), SARS-CoV-2 (or 2019-novel coronavirus, 2019-nCov). wherein the ACE inhibitor is lisinopril, and wherein the type I angiotensin II receptor blocker is losartan.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0010] FIG. 1: Potential therapeutics for environment- and virus-induced lung injury based on balancing the Renin-angiotensin system (RAS).

[0011] FIG. 2: The application of recombinant human ACE2 proteins to inhibit and treat SARS-CoV-2 infection.

[0012] FIG. 3: The application of a lentiviral vector to stably over-express ACE2 in human cells.

DETAILED DESCRIPTION

[0013] As shown in FIG. 1, Activation of the RAS cascade involves Renin and cleaves angiotensinogen to generate Ang I, which is then converted to Ang II by ACE. Ang II binds to its receptors (AT1 and AT2) to exert local and systemic effects such as vasoconstriction and promotion of the release of aldosterone. ACE2 functions as a counter-regulatory enzyme for balancing responses initiated from ACE. ACE2 hydrolyses Ang I and Ang II to generate Ang-(1-9) and Ang-(1-7). Ang-(1-7) binding to the MAS receptor antagonizes Ang II-mediated actions. As an example, viruses such as SARS-CoV and 2019-nCoV use ACE2 as the entry receptor. Inhibition of ACE2 expression or downregulation of surface ACE2 by these coronaviruses may disrupt function balances between ACE/ACE2, which may be alleviated by different approaches (FIG. 1. from A, B, C, to D).

[0014] There are several potential therapeutic approaches that can be tested or developed (FIG. 1). First, therapies to increase ACE2 expression can be developed, through direct injection of recombinant ACE2 protein, which has been shown to protect mice from severe acute lung injury (Imai et al., 2005), and by delivering therapeutic vectors expressing high levels of ACE2 directly into lung tissues to overcome virus-induced ACE2 deficiency. Second, certain ACE inhibitors such as lisinopril may be used to balance the ACE/ACE2 function. In addition, therapeutic Ang-(1-7) heptapeptide may be delivered to activate its receptor MAS and to counteract the activities of Ang II. Furthermore, drugs blocking Ang II receptors may also be tested. In particular, the type I, but not type II, Ang II receptor has been shown to promote disease pathogenesis by inducing lung oedemas and impairing lung function (Imai et al., 2005). Thus, a type I Ang II receptor blocker such as losartan could be tested for alleviating SARS-CoV-2-induced lung injury (FIG. 1).

[0015] The use of recombinant human ACE2 proteins to block SARS-CoV-2 (or 2019-nCoV) virus infection of human cells. As shown in FIG. 2, HEK293T(ACE2/TMPRSS2) cells stably expressing human ACE2 and TMPRSS2 were used as the target cell for SARS-CoV-2 viral particle infection. The target cell was not infected (Cell only) or infected with a hybrid SARS-CoV-2 virus like particles (+SARS-CoV-2) carrying a luciferase reporter, as described previously (Hetrick et al., 2020). Target cells were also pre-treated with recombinant human ACE2 protein monomer (extracellular domain of ACE2, amino acid 1-615) or with ACE2 protein dimer (extracellular domain of ACE2, amino acid 1-720), and then infected in the presence of these ACE2 proteins in different concentrations (133 ug/ml to 5.3 ug/ml, ACE2 monomer; 121 ug/ml to 3.8 ug/ml, dimer). Following infection for 18 hours, viral entry and gene expression was quantified by luciferase assay. FIG. 2 shows ACE2 protein dosage-dependent inhibition of the infection of target cells by the hybrid SARS-CoV-2 particles.

[0016] The use of a lentiviral vector to over-express ACE2 in human cells. As shown in FIG. 3, a lentiviral vector expressing the full-length human ACE2 gene (pLenti-hACE2-puro) was used to assemble a lentiviral particles, vLenti-hACE2-puro, which was then used to transduce human HEK293T cells. Following transduction, cells were selected in puromycin (1 μg/ml) for cells stably expressing ACE2; the puromycin gene was co-expressed with the ACE2 gene from pLenti-hACE2-puro. Cells that stably transduced with vLenti-hACE2-puro were analyzed for ACE2 over-expression by surface staining of ACE2 with a commercial anti-human ACE2 antibody and flow cytometry. Shown is the over-expression of ACE2 in cells stably transduced with vLenti-hACE2-puro (HEK293T-ACE2), in comparison with the un-transduced HEK293T cells (HEK293T).

[0017] Illustrative Examples are presented below. They are exemplary and non-limiting.

Example 1: Potential Therapeutics for Environment- and Virus-Induced Lung Injury Based on Balancing the Renin-Angiotensin System (RAS)

[0018] As exemplified in FIG. 1, SARS-CoV-2-induced lung injury can be inhibited by balancing the RAS system.

[0019] As exemplified in FIG. 2, SARS-CoV-2 virus entry and infection of target cells can be blocked by the application of recombinant ACE2 proteins to inhibit viral infection of ACE2+ target cells, thus reducing virus-induced lung injury.

[0020] As exemplified in FIG. 3, the use of a lentiviral vector to over-express ACE2 to increase expression of ACE2 in cells.

Experimental Procedures

[0021] Inhibition of Hybrid SARS-CoV-2 Reporter Virus Infection by Recombinant Human ACE2 Proteins

[0022] HEK293T(ACE2/TMPRSS2) cells were seeded into a 12-well tissue culture plate (2×10.sup.5 cells per well) in 1 ml cell culture medium (DMEM+10% heat-inactivated FBS, lx penicillin-streptomycin). Cell were grown overnight at 37° C.

[0023] Dilution of recombinant human ACE2 proteins

[0024] Dilution of purified recombinant human ACE2 monomeric protein (containing ACE2 extracellular domain, amino acid 1-615).

[0025] A—ACE2 monomer protein concentration, 2,000 μg/ml

[0026] A1—1:5 dilution—took 30 μl A+120 μl culture medium, concentration: 400 μg/ml

[0027] A2—1:25 dilution, took 30 μl A1+120 μl culture medium, concentration: 80 μg/ml

[0028] A3—1:125 dilution, took 30 μl A2+120 μl culture medium, concentration, 16 μg/ml

[0029] Dilution of purified recombinant human ACE2 dimeric protein (containing ACE2 extracellular domain, amino acid 1-720)

[0030] B—ACE2 dimer protein concentration, 1400 μg/ml

[0031] B1—1:5 dilution—took 42 μl B+120 μl culture medium, concentration: 363 μg/ml

[0032] B2—1:25 dilution, took 30 μl B1+120 μl culture medium, concentration: 72 μg/ml

[0033] B3—1:125 dilution, took 30 μl B2+120 ul culture medium, concentration, 14.5 μg/ml

[0034] 100 μl of A1, A2, A3, B1, B2, or B3 were mixed with 100 μl of hybrid SARS-CoV-2(Luc) reporter virus, individually. As a control, 100 μl cell culture medium was mixed with 100 μl of hybrid SARS-CoV-2(Luc) reporter virus. Each resulting mixture was incubating at 37° C. for 30 minutes. Samples were labeled as A1+virus, A2+virus, A3+virus, B1+virus, B2+virus, B3+virus, and C+virus.

[0035] For infection of cells, 900 μl of culture medium were removed from each well of the 12-well plate, and 100 μl of medium were left in each well. The 200 μl of the mixture of A1 to B3 plus virus (prepared in step 3) were added. For control, the 200 μl of the mixture of medium plus the reporter virus were added. Cells were infected for 2 hours at 37° C. Infected cells were washed once, and then 1 ml fresh medium was added. The infected cells continued to culture for 18 hours.

[0036] Analysis of viral infection by luciferase assay. The infected cells were harvested and placed in a 1.5 ml micro-centrifuge tube. The cells were pelleted by centrifugation for 1 minute in a microfuge. The cell pellet was washed once with 1×cold PBS and then the cell pellet was resuspended in 100 μl Luciferase Assay Lysis Buffer. Luminescence was measured by using GloMax Discover Microplate Reader. The results are shown in FIG. 2.

[0037] The Use of Lentiviral Vector to Over-Express ACE2 in Human Cells

[0038] Assembly of lentiviral particles for ACE2 over-expression:

[0039] HEK293T cells were seeded into 10-cm tissue culture dish (3×10.sup.6 cells per dish) in 10 ml cell culture medium (DMEM+10% heat-inactivated Fetal Bovine Serum, lx penicillin-streptomycin). Cells were grown overnight at 37° C.

[0040] The next morning, cell culture medium was replaced with serum-free DMEM medium, 9 ml per dish.

[0041] The transfection mixture was prepared with 10 μg of pLenti-hACE2-puro vector, 8 μg of pCMVΔR8.2, and 2 μg of pHCMV-VSV-G. Serum-free DMEM medium was added to the DNA mixture to a total volume of 500 μl.

[0042] Transfectin™ (from Virongy LLC) was used for DNA transfection. 45 μl of Transfectin™ was added into 455 μl of Serum-free DMEM medium for a final volume of 500 μl. For transfection, 20 μg of DNA mixture (in 500 μl serum-free DMEM medium) prepared above was mixed with 500 μl Transfectin™ mixture. The combined solution was vigorously mixed and then added into HEK293T cells for 6 hours. The transfection supernatant was removed, and replaced with 10 fresh DMEM medium with 10% heat-inactivated Fetal Bovine Serum. Cells were cultured for 48 hours at 37° C.

[0043] The viral particles were harvested at 48 hours post cotransfection and were named as vLenti-hACE2-puro.

[0044] Transduction of HEK293T cells with vLenti-hACE2-puro for ACE2 over-expression:

[0045] HEK293T cells were cultured in 6-wells plate by seeding 2×10.sup.6 cells and grown overnight at 37° C.

[0046] 500 μl of vLenti-hACE2-Puro viral particles were seeded for infection of cells for overnight.

[0047] 2 ml fresh medium were added the next day and cultured for 2 more days.

[0048] Puromycin at 1 μg/ml were added. The cells were allowed to continue to culture, and the medium was changed every two days with fresh puromycin (1 μg/ml) being added into the medium.

[0049] The puromycin-resistant cells were transferred to T25 flasks, and then, to T75 flasks to grow cells to large volumes. Following selection, all cells were puromycin-resistant.

[0050] For surface staining analysis of ACE2 over-expression, both the parental HEK293T cells and the vLenti-hACE2-puro stably transduced HEK293T(ACE2) cells were used for staining with an anti-human ACE2 antibody. Cells were analyzed by flow cytometry as shown in FIG. 3. The vLenti-hACE2-puro transduced cells had higher ACE2 surface expression.

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