mRNA-enhanced cell therapy and cardiovascular regeneration

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mRNA-enhanced cell therapy and cardiovascular regeneration

mRNA-enhanced cell therapy and cardiovascular regeneration.  During the COVID-19 pandemic, mRNA has become an important biomolecule for global therapeutic development.

Synthetic in vitro transcription (IVT) mRNA can be designed to mimic naturally occurring mRNA and can be used as a tool to target “non-treatable” diseases. Recent advances in the field of RNA therapy have solved the inherent challenges of this drug molecule, and this approach is now used in a variety of treatment modalities, from cancer immunotherapy to vaccine development.

In this review, we discussed the use of mRNA to generate or enhance stem cells for the purpose of cardiovascular regeneration.

1. Introduction

The progress of the biopharmaceutical industry has accelerated in the global competition to respond to the COVID-19 pandemic. Most notably, mRNA vaccines have inspired this field, and the speed of light has produced new therapeutic molecules. For example, within 42 days after Chinese scientists announced the SARS-CoV-2 sequence in January 2020, Moderna sent its RNA vaccine candidates to the National Institute of Allergy and Infectious Diseases for preclinical testing.

By April 2020, Moderna has launched its first clinical trial. Less than 8 months later, Moderna will seek emergency use authorization for the vaccine, and the Phase III trial has shown 95% effectiveness and excellent safety. Of the 236 COVID-19 vaccines under development, 29 are mRNA-based.

The first two of all vaccines that have completed Phase III clinical trials (BNT162 from Pfizer and MRNA1273 from Moderna) fall into this category. These vaccines will be the first mRNA therapies to enter the market. The development speed, high efficiency and safety of mRNA vaccines have attracted people’s attention to the prospects of mRNA therapy.

Although most of the drugs approved by the FDA are small-molecule drugs, these drugs have limitations in the scope of “available drugs” for diseases. In contrast, the scope of mRNA is almost unlimited, because this biological software can be quickly modified to encode any therapeutic protein or antigen of interest. In addition, with the advancement of administration methods, pharmacokinetics and pharmacodynamic properties, enhanced efficacy and stability, and reduced immunogenicity and production costs, mRNA therapy has almost unlimited potential.

mRNA therapy has advantages over contemporary small molecule, protein or DNA-based therapies. For example, it is difficult to generate small molecules that allosterically enhance the activity of defective enzymes. Compared with DNA-delivered gene carriers, RNA has biological activity in both dividing and non-dividing cells, and does not need to enter the nucleus to produce therapeutic effects. In addition, using standard mRNA, there is no risk of altering the host genome, etc.

The mRNA-based approach facilitates a variety of treatment modalities, including (a) replacement therapy to compensate for defective genes/proteins, or to provide therapeutic proteins (b) vaccination, in which mRNA encoding a specific antigen is injected to trigger protection Sexual immunity (c) Cell therapy, including transfection of mRNA into cells in vitro to therapeutically enhance cell survival, proliferation and/or function (d) Use mRNA to produce new monoclonal antibodies (e) Gene editing, mRNA It is used to express an enzyme that can edit and correct defective genes that cause disease.

Advances in mRNA design, production, and delivery have led to the exploration of mRNA therapy in different fields, such as immunotherapy for cancer and infectious diseases, the production of growth factors, the production of engineered mesenchymal stem cells (MSCs), and regenerative medicine.

2. Using mRNA to produce iPSCs for stem cell therapy

In 2006, Yamanaka and colleagues demonstrated that by using retroviral vectors to ectopically express a specific set of transcription factors Pou5f1, Sox2, Klf4, and c-Myc (OSKM), terminally differentiated adult somatic cells can be reprogrammed to generate induction Competent stem cells (IPSC). These iPSCs are highly similar to embryonic stem cells (ESC) in their ability to self-renew and differentiate into all three germ layers.

Therefore, human iPSCs can be used as a substitute for human ESCs, thereby avoiding potential ethical issues. This groundbreaking discovery is a revolution in the field of regenerative medicine. Patient-specific iPSCs produced from somatic cells can be differentiated to understand the pathobiology of genetic diseases.

In addition, differentiated derivatives of iPSCs can be used as therapeutic cells. For example, iPSCs produced by patients with genetic diseases (such as cardiomyopathy caused by muscular dystrophy) can be edited in vitro. These iPSCs can be differentiated into therapeutic cells and transplanted into patients with minimal risk of genetic incompatibility or immune rejection.

However, the viral vectors (retroviral, lentiviral or adenoviral vectors) used to produce iPSCs carry the risk of genome integration and limit the clinical application of such iPSCs. Therefore, several non-integration methods have been developed, including Sendai virus, cell permeation recombinant protein, non-integrating plasmid or epitope DNA. Although the risk of genome insertion in these methods is small, the efficiency of iPSC generation is very low. Subsequently, iPSCs were generated using mRNAs encoding POU5F1, SOX2, LIN28A, and NANOG or mRNA encoding Yamanaka factor. The characteristics of the iPSCs generated by the two mRNA-based methods indicate that the overall transcription characteristics of human ESCs are more consistent than retrovirus-derived iPSCs. SNP analysis showed that iPSCs derived from retroviral vectors have more mutations than iPSCs derived from mRNA.

These observations indicate that in clinical applications, mRNA-based methods are safer than retrovirus-derived iPSCs. Although the mRNA-based method produced a genetically modified IPSC with a reasonable reprogramming efficiency (4%), the protocol required transfection for 2 weeks per day. Recent research has simplified and optimized mRNA-based reprogramming schemes. However, once produced using mRNA technology, such iPSCs can be differentiated into clinical grade cardiomyocytes using standard differentiation protocols. In addition, mRNA can be used to accelerate the differentiation of iPSCs into desired derivatives. For example, mRNA encoding ETV2 has been used to generate iPSC-derived endothelial cells with high efficiency (90%).

3. Use mRNA to directly produce or enhance therapeutic cells

It is also possible to use mRNA to directly generate therapeutic cells and/or enhance their proliferation, survival or function. For example, mRNA encoding reprogramming or differentiation factors can be transfected into somatic cells derived from readily available somatic cells (eg, skin fibroblasts) to directly generate cardiovascular cells in vitro. In theory, these cells can be injected directly back into the patient’s body or integrated into a biocompatible scaffold. For example, by using retroviral methods to overexpress the major regulators of the cardiomyocyte lineage (ie, Gata4, Mef2c, and Tbx5), fibroblasts have been directly transdifferentiated into cardiomyocytes in vitro and in vivo.

Similarly, we and others have used viral vectors that overexpress the main regulators of the endothelial cell lineage (such as ETV2, FLI1, GATA2, and KLF4) to transdifferentiate fibroblasts into induced endothelial cells. Compared with viral vectors, mRNA encoding the major regulators of the cardiovascular lineage can be used to achieve therapeutic transdifferentiation in an unintegrated manner, causing fewer safety issues. In fact, using mRNA to encode these endothelial transcription factors, we successfully transdifferentiated human fibroblasts into endothelial cells, whose transcription and function are highly similar to real human endothelial cells.

Although the optimization of mRNA structure and vector is still necessary for the introduction of these therapies into clinical practice, with the emergence of proof-of-concept studies, they have great prospects. For example, the use of mRNA differentiation cocktails for cardiac reprogramming of human mesenchymal stromal cells has recently been demonstrated. Similarly, the overexpression of human vascular endothelial growth factor A (VEGFA) and mRNA promotes the endothelial specificity of human ISL1+ progenitor cells and their implantation, proliferation and survival in the body.

Mesenchymal stromal cells (MSCs) can be obtained from different sources, such as umbilical cord, bone marrow, liver, adipose tissue, and various dental tissues. These cells have the ability to self-renew, differentiate into different cell lines, migrate to the injured site, and secrete proteins that reduce inflammation, promote angiogenesis and tissue repair. Clinical trials are ongoing to evaluate the benefits of autologous bone marrow mesenchymal stem cells for patients with ischemic syndrome or cardiomyopathy. Due to the heterogeneity of the quality of biological products, these tests have become more difficult. The strategy to improve the efficacy and uniformity of cell therapy is through mRNA enhancement. In fact, synthetic mRNA is being applied to the engineering of MSCs. Synthetic mRNA regulates the migration characteristics of MSCs by temporarily expressing homing protein on the cell surface. In this way, mRNA-modified MSCs can target vascular inflammation.

C-X-C motif receptor 4 (CXCR4) is a chemokine receptor that binds to matrix-derived factor-1 (CXCL12) that is expressed with high affinity at sites of inflammation. MSCs transfected with mRNA CXCR4 showed improved cell migration to CXCL12 in transwell experiments, indicating that mRNA-mediated overexpression of chemokine receptors can trigger the transient initiation of chemotaxis. Therefore, by synthesizing mRNA to promote the migration of MSCs, the regeneration ability of damaged tissues can be enhanced.

4. Inflammatory signals in nuclear reprogramming and transdifferentiation

The induction of pluripotency, and the transdifferentiation of one somatic cell into another, requires inflammatory signals. For example, although the Yamanaka factor OSKM provides the direction of transcription, we now know that retroviral vectors also activate inflammatory signals to increase chromatin accessibility, thereby allowing the Yamanaka factor to act on the promoter sequences of the gene network required for pluripotency. Whether using viral vectors or mRNA to induce pluripotency, the activation of cell-autonomous inflammatory signals is necessary for nuclear reprogramming to pluripotency.

The inflammatory signals required for nuclear reprogramming are mediated by pattern recognition receptors (PRRs), which can sense pathogen-related molecular patterns (PAMPs) or damage-related molecular patterns (DAMPs). Exogenous mRNA activates toll-like receptors (TLRs) 3 and 7. Stimulating these TLRs can trigger inflammatory signaling pathways and activate NFkB, IRF-3 and IRF-7, the latter can induce genes encoding inflammatory cytokines and chemokines. In addition, these signaling pathways cause overall changes in the expression of epigenetic modifiers, changing the balance between chromatin activators and inhibitors. For example, inflammatory signals up-regulate several members of the histone acetyltransferase (HAT) family, while members of histone deacetylase (HDAC) are down-regulated.

In addition, this inflammatory signal leads to post-translational modification of epigenetic modifiers, supporting the possibility of an open chromatin state. The expression of inducible nitric oxide synthase (NOS2) increases NFKB and transfers to the S-nitroacylated ring 1A of polyclonal B inhibition complex 1 (PRC1), and binds to the nucleus. The S-nitration of PRC1 removes it from chromatin and eliminates this inhibitory effect. Similarly, NuRD complexes are also S-nitroated through this inflammatory signaling process, preventing its deacetylation and inhibiting chromatin. In addition to reactive nitrogen species, it seems that the instantaneous generation of reactive oxygen species is required in order to reprogram effectively.

Finally, the metabolic conversion from oxidative phosphorylation to glycosylation is essential for transgenes. Exogenous mRNA triggers the glycation switch, which is related to the export of citric acid in the mitochondria. In the nucleus, citric acid is converted to an increase in acetyl-CoA, thereby providing a substrate for histone acetylation. The adversarial nature of this process eliminates the nuclear reprogramming required for phenotypic switches. Although most discussions on mRNA therapeutics emphasize the need to reduce the immunogenicity of the construct, it is clear that when the cell phenotype needs to be changed, exogenous mRNA requires a certain level of inflammatory signals to perform its effect. The role of inflammatory signals in promoting chromatin opening is essential for transcriptional activators to enter their consensus sequence. On the other hand, excessive activation of inflammatory signals may interfere with the required phenotypic switch. In fact, in the process of nuclear reprogramming to pluripotency, there seems to be a “golden zone” for optimal inflammatory signaling.

5. Use mRNA to reverse cardiovascular aging

One of the main determinants of cell aging is telomere erosion. The telomeres of somatic cells become shorter with each division. This is due to the “end duplication problem”. Oxidative stress accelerates this process. When the cell approaches the Hayflick limit, the telomere length reaches a critical threshold, triggers the DNA damage response, activates the TP53/CDKN1A pathway, and leads to cell cycle arrest, senescence and degeneration. Telomerase is a protein that reverses this process by lengthening telomeres. This protein is present in pluripotent stem cells and to a certain extent in adult stem cells, explaining the increased ability of these cells to replicate. Telomerase is generally not present in somatic cells, but can be reactivated in rapidly proliferating immune cells.

Our interest in using mRNA encoding human telomerase (TERT) as a treatment for vascular aging comes from our and others’ research in the past 25 years. These studies have shown that vascular aging is related to endothelial diseases that promote the process of atherosclerosis. For example, Chang and Harley measured the telomere length of human iliac arteries and mammary arteries and found that telomere length decreases with age. In addition, the telomeres of the iliac arteries are shorter in every age group (the iliac arteries are more prone to atherosclerosis). We have proven that aging human endothelial cells produce less nitric oxide (NO), more superoxide anions (O2-), synthesize more adhesion molecules, and have stronger adhesion to monocytes. And the multiplication ability is reduced, which is consistent with the fluid shear stress. These properties promote vascular inflammation and atherosclerosis. In contrast, when we overexpress telomerase using retroviral vectors, telomere lengthening is associated with the reversal of age-related endothelial diseases and the restoration of endothelial cell proliferation and function.

However, retroviral integration of telomerase in human cells has raised concerns about unregulated growth. Therefore, we use mRNA TERT to transiently express telomerase, increase telomere length, enhance replication ability, and reverse the signs of aging in human somatic cells. After each transfection, the telomerase activity lasted no more than 72 hours (by TRAP analysis). However, 1-3 treatments increased telomere length, population doubling, and decreased expression of senescence markers, β-galactosidase (β gal) was found in fibroblasts, endothelial cells, and myoblasts, although the cells eventually grew Stable (Figure 1).

Unexpectedly, transient transfection with TERT can produce long-term cellular benefits. On this basis, we have developed a new mRNA therapeutic agent (codon optimization, UTR modification, HPLC purification of mRNA telomerase in lipid nanoparticles) for the treatment of endothelial diseases associated with vascular aging. Our treatment has higher stability and less immunogenicity to increase transcription and reduce toxicity. Because endothelial disease is the basis of many cardiovascular diseases, as well as other age-related diseases, such as vascular dementia, peripheral arterial disease, nephrosclerosis, pulmonary fibrosis and impaired wound healing, the correction of endothelial lesions caused by aging is expected to reduce or reverse many diseases and disorders related to aging.

As an accelerated aging model, we studied cells from children with Hutchison Whampoa Progeria Syndrome (HGPS). We have observed that the use of mRNA-TERT transient transfection of HGPS child-derived cells can increase telomere length, restore replication ability, reduce the expression of senescence markers, and improve the cell function of fibroblasts, iPSC-derived endothelial cells and vascular smooth muscle cells (figure 2). Interestingly, mRNA-TERT reduced the level of progerin and improved the morphology of the cell nucleus. In addition, we found that the mRNA-hTERT treatment of HGPS cells is superior to the current treatment using the farnesyltransferase inhibitor lonafarnib through assessment of senescence markers, proliferation index and nuclear morphology.

One warning of telomerase treatment is the possibility of cancer. In approximately 85% of cancers, human telomerase is activated. However, using mRNA to express telomerase transiently is unlikely to increase the risk of cancer. We have a lot of data showing that telomerase activity lasted less than 72 hours after transient transfection with tertmRNA. In addition, although the replication ability of cells has been improved, the growth curve of TERT-treated cells has a normal pattern, that is, there is a normal logarithmic growth phase, reaching a stable phase, that is, TERT-treated cells are not immortalized.

In fact, this treatment may reduce the risk of cancer: Senescent cells with shorter telomeres are more likely to experience a “crisis,” leading to DNA damage responses, chromosome fusion, and reactivation of telomerase, which can lead to cancer. The extension of telomeres can prevent this aberration. It is worth noting that our preliminary studies have shown that mRNA telomerase treatment reverses the markers of DNA damage in HGPS and is expected to reduce the risk of tumor occurrence.

6. Use mRNA to promote cardiovascular regeneration

In addition to modifying therapeutic cells in vitro, mRNA can also be delivered directly to tissues to produce therapeutic effects. This was first confirmed in 1992, when Jirikowski and his colleagues injected synthetic mRNA encoding antidiuretic hormone (vasopressin) into the hypothalamus of rats with vasopressin gene defects.

These animals suffer from diabetes insipidus, which is characterized by difficulty in collecting urine and excreting large amounts of diluted urine. In these animals, injection of vasopressin mRNA into the hypothalamus can induce the synthesis of vasopressin protein and temporarily reverse the disease.

Since then, the feasibility of using mRNA to replace defective or missing proteins for therapeutic purposes has been demonstrated in multiple studies and various tissues. Although most mRNA-based therapies are still in preclinical development, more and more candidate methods have won first place in human trials.

Zangi et al. first reported the feasibility of direct intramyocardial injection in 2013. In this basic work, vegfamrna was injected into the ischemic area of ​​the mouse myocardium when the coronary arteries were ligated. The local increase of VEGFA induces the expansion and directed differentiation of endogenous cardiac progenitor cells. In addition, this intervention significantly improved the heart function of experimental myocardial infarction (MI) mice and increased their long-term survival rate. It is worth noting that, unlike mRNA, VEGFA encoding plasmid DNA significantly reduces the survival rate of MI animals. This unexpected finding may be related to the time difference of VEGFA expression, because prolonged exposure to VEGFA expressed by plasmid DNA is associated with abnormal vascular permeability and myocardial edema.

Carlsson et al. subsequently confirmed the effect of VEGFA mRNA on heart regeneration after myocardial infarction in large animal models. In this study, myocardial infarction was induced by permanent ligation of the middle anterior descending branch of the left coronary artery of mini-pigs, and naked mRNA was injected into the infarct area and peri-infarct area 7 days after the first operation. Two months after the injection, the left ventricular ejection fraction, contractility, and myocardial compliance were significantly improved. In addition, in the heart treated with vegfamrna, the blood vessel density in the area around the infarction increased and myocardial fibrosis decreased. It is worth noting that in this study, the toxicity of mRNA was also evaluated, and at 24 hours after injection, whether it was injected intracutaneously or intravenously to rats and cynomolgus monkeys, serum levels of pro-inflammatory cytokines were not increased.

These encouraging data prompted AstraZeneca (AZD8601) to cooperate with Moderna to conduct the first clinical trial of mRNA therapy for cardiac regeneration. The EPICCURE study (NCT03370887) is a randomized, placebo-controlled, double-blind, multi-center, phase 2a clinical trial to study the safety and effectiveness of epicardial injection of VEGFA mRNA. The inclusion criteria specified patients with stable coronary artery disease and moderately reduced left ventricular ejection fraction who are undergoing coronary artery bypass graft surgery. The study is currently under registration and is expected to be completed in early 2023. Before the start of EPICCURE, a randomized, double-blind, placebo-controlled phase 1 study was conducted on male patients with type 2 diabetes, in which AZD8601 was injected intracutaneously into the forearm skin as a single increasing dose, and was safe for 6 months Follow up. The only treatment-related adverse event observed in the experiment was a mild injection site reaction, and the local skin blood flow increased significantly within 7 days after mRNA injection, and was related to the VEGFA protein concentration in the skin dialysate collected in this area. There are similar findings in preclinical studies, where VEGF-amrna also promotes the healing of diabetic wounds.

Although VEGFA is currently the most advanced therapeutic candidate for cardiovascular regeneration mRNA, the results of preclinical studies of some other constructs targeting different molecular pathways have been reported. For example, Chen et al. demonstrated that the transcriptional co-activator yes-related protein (YYIAP1) mRNA improves myocardial outcome after ischemia-reperfusion (IR) injury in mice. Yyiap1mrna can significantly reduce the innate immune inflammatory response of the injured myocardium and the survival rate of myocardial cells. After 4 weeks, the heart function is improved and the hypertrophy remodeling is inhibited.
Zangi et al. also demonstrated the feasibility and benefits of using mRNA to manipulate the IGF-1 signaling pathway. However, their research shows that although Igf1 receptor stimulation can increase the survival rate of cardiomyocytes and cardiac progenitor cells, it may also promote the formation of adventitia in the center of the injured heart.

7. Future Outlook

With the improvement of mRNA structure to improve its stability, enhance translation, and promote delivery to target tissues, the field of RNA therapeutics is growing exponentially and developing toward clinical applications.

Recently, the success of an mRNA vaccine against SARS-CoV-2 has aroused great interest, which will further accelerate the development of this exciting medical frontier. RNA therapy is a disruptive technology, because small biotech startups and academic groups can quickly develop new personalized structures.

(source:internet, reference only)

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