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A Functional, Mature In vivo Human Ventricular Muscle Patch for Cardiomyopathy

Periodic Reporting for period 2 - 5D Heart Patch (A Functional, Mature In vivo Human Ventricular Muscle Patch for Cardiomyopathy)

Reporting period: 2019-06-01 to 2020-11-30

The human ventricular myocardium has a limited capacity for regeneration, most of which is lost after 10 years of age. As such, new strategies to generate heart muscle repair, regeneration, and tissue engineering approaches during cardiac injury have been a subject of intense investigation in regenerative biology and medicine. Given the need to achieve coordinated vascularization and matrix formation during tissue engineering of any solid organ, the assumption has been that the formation of an intact 3D solid organ in vivo will ultimately require the addition of vascular cells and/or conduits, as well as biomaterials and/or a de-cellularized matrix that will allow alignment and the generation of contractile force. The complexity of adding these various components to achieve the formation of a functional solid organ has confounded attempts to reduce this to clinical practice. Although human pluripotent stem cells (hPSCs) hold great promise, to date, it has not been possible to build a pure, vascularized, fully functional, and mature 3D human ventricular muscle organ in vivo on the surface of a heart in any mammalian system. The generation of a fully functional, mature in vivo 3D ventricular heart muscle patch from hPSCs would be a major advance in tissue engineering and regenerative therapeutics. However, it has not yet been possible to drive hPSCs derived cardiomyocytes to recapitulate the discrete steps of vascularization, matrix formation, assembly, and maturation required for in vivo human ventriculogenesis. Accordingly, the central objective of this proposal is to optimize the generation of a large scale, fully functional human ventricular muscle patch in vivo through the self-assembly of purified human ventricular progenitors and the localized expression of defined paracrine factors that drive their expansion, differentiation, vascularization, matrix formation, and maturation.

The project is designed to address several important advances on each of the proposed aims. These are noted below:

1. In order to use this technology as a potential path forward for repairing diseased cardiac function in patients, we must uncover mechanisms that can enhance graft size, function and maturation. To accomplish this, we have built a paracrine factor library using small, chemically modified mRNA compounds, which have already been shown to be safe in patients (Gan et al., 2019). By fusing these two technologies we believe we will be more capable of improving heart function in patients with diseased hearts suffering from weakened pumping capacity. In order to identify the absolute best compounds to fuse with the HVP cells, we must first perform a number of in vitro (cell based experiments in culture dishes) screens. Once we nominate the top candidates to fuse with HVP technology, we will employ small animal studies to test the efficacy of the stem cell-mRNA based technology as a potential therapy to move forward into larger pre-clinical and clinical studies.

2. One other aspect of interest consists in exploring the possibilities of using the HVP technology to model human cardiac genetic disorders. In this case we decided to focus on phospholamban related cardiomyopathies due to its strong association to a specific human phenotype and the clear scientific gap present in our understanding of human calcium mishandling. Previously published studies show indications of possible differences in between murine and human calcium cardiac physiology and pathophysiology, emphasizing the importance in developing new relevant human disease modeling tools to establish relevant translatable studies. In order to explore these possibilities, we propose to establish genetically modified human embryonic stem cell lines and to generate HVPs from these lines. In order to study the effect of phospholamban mutations we will also generate modified mRNA of these mutants in order to efficiently express the mutant proteins in the HVP patches. We believe that by doing so, we will be able to uncover mechanistic insights on the development of phospholamban related cardiomyopathies in human.

3. Towards generation of a human heart patch, we have employed a novel non-human primate ex vivo heart patch system developed by collaborators at the Technical University of Munich (TUM). These studies have identified the ability of HVPs to migrate directly to the site of injury, to expand, differentiate, and also to prevent fibrosis of the injured tissue. These studies have laid the ground work for the potential clinical tractability and potential of the HVPs. Given the pivotal role of this model system, we have proposed an amendment to our original application to allow support of the collaboration with TUM via our ERC grant, which has been formally submitted.
"1.The HVP study has developed an ex vivo Non Human Primate (NHP) heart regeneration model, in which we took strips of (viable and functioning) NHP ventricular muscle and continuous record their force generation and calcium transients for a few weeks in a co-culture system seeded with HVPs. Subsequently, the native NHP cardiomyocytes would die off after 2 weeks, and the HVPs have the ability to regenerate the muscle; not only on the histological but also the functional level. Moreover, we have established an ex-vivo injury model in which we use radio frequency ablation to induce injury only at the cellular level and not on the matrix. With that model, we have identified several key steps in the process of regeneration at the cellular level (i.e. the HVPs have the capacity to migrate to the injury site, and replace the injured muscle). Thereafter, we have demonstrated that HVPs have the unique ability to prevent cardiac fibrosis via cardiac fibroblast repulsion. Single cell analysis revealed the molecular pathways responsible, and validated by mechanistic in vitro studies. Furthermore, we have now confirmed our in vitro, ex vivo results with in vivo pig study. We plan on performing further in depth study with clinically relevant porcine model to examine potential arrythmogenesis.

2.In order to fully understand the key drivers that regulate heart development and growth, our team explored and identified pertinent genes and proteins in the developing human fetal heart (Sahara et al., 2019). The most interesting identified growth factors have been made into modified mRNA compounds for exploratory research purposes. Next, we have explored several different mechanisms for delivering these compounds into the HVP cells. One mechanism involves combining the molecules with a ""carrier"" so the molecules can penetrate inside the cells and the other method relies on quickly ""pulsing"" the cells with voltage allowing the molecules to be introduced inside the cells. Once the mRNA molecules are delivered inside the cell, we can begin to assess the effects these molecules have at enhancing cell proliferation, cell growth, and strengthening the commitment of these cells to becoming heart muscle cells. We are now in the process of screening these compounds, where we will score them independently in each group. Once we have scored the compounds, the mRNA molecules that are best ranked will be combined with HVPs and implanted into diseased animal models. Here, we can examine whether the survival, growth, maturation and vascularization of the heart muscle patches are enhanced by this novel combinatorial approach. We have preliminary data now that highlights combining VEGF mRNA with our HVPs can enhance the vascularization (increased blood flow) of the muscle patch in an in vivo setting, in non-diseased animals. Much more work is needed to show how this treatment is capable of maturating the patch and enhancing long-term survival and function of the muscle patch. What will be more interesting is to combine VEGF mRNA with several of our new compounds and explore the long-term survival and function of the muscle graft in the diseased setting.

3.For the usage of HVPs as a human disease model, we have established genetically edited human embryonic stem cell lines of phospholamban heterozygous and homozygous knockout. We have also generated a library of modified mRNAs of PLN, mutant PLNs and different tagged versions of these constructs. All of these tools have been thoroughly quality controlled and we are now in process of obtaining preliminary data on their effects on calcium regulation in heart cells derived from human stem cells.
We have also generated and optimized modified mRNA of Cas9 and obtained high efficiencies of genetic mutations of the targeted PLN sequence in human and mouse embryonic stem cells."
1.Many novel therapies for treating cardiac disorders can be grouped into 1 of 2 categories, cell-based therapies or cell-free therapies. To date, many clinicians and scientists stand on one side of this coin, in favor of either cell-based therapies or in favor of cell-free (molecule/chemical) therapies. What is special about our research approach is that we aim to combine cell therapies with small molecules to enhance cardiovascular reparative therapies. This concept is quite novel. As we have not yet elucidated the mechanisms of our novel compounds, it is difficult to make any predictions regarding the expected results. We can however, envision that a vascularized muscle graft should enhance long-term survivability and function of the naïve HVP graft. Many pre-clinical studies in large animals have shown transplanting mature cardiac cells in diseased heart models fail to significantly enhance cardiac function. One plausible explanation to these results is because many of the injected cells are pumped out of the heart following transplantation. The remaining cells may maturate into residual small islands of new heart muscle but are incapable of providing a strong global functional effect in the heart. As these islands of newly formed muscle are poorly vascularized, they fail to survive long-term. We predict that fusing our cardiac progenitor cells with VEGF-mRNA will enhance blood flow to the muscle patch, thus increasing oxygenation and nutrients, which are important to the long term stability and function of the graft.

2.One other aspect of the HVPs is its potential to further develop human cardiac disease modeling. Many genetic disorders linked to cardiac diseases have been modeled in the mouse. Those models despite being useful, do possess strong limitations due to basal differences in between mouse and human cardiac physiology. Therefore, in order to push forward our knowledge in this field and increase translatability of our potential findings we proposed the combination of state of the art generation of human ventricular patches through our HVP technology, the flexibility of their genetic manipulation through CRISPR and the selectivity of mutant protein expression through our modified mRNA library