Development of iPSC-derived models and cell-penetrating peptides towards therapeutic targeting of vascular anomalies

The cardiovascular system maintains our body homeostasis and keeps body systems in balance to function correctly. This is achieved by the heart pumping blood with oxygen and nutrients through vessels which transport blood to all tissues and removes waste products from tissues. Vascular anomalies (VAs) are malformations of either blood, or lymphatic vessels. VAs usually arise during embryonic development, as a consequence of inherited or somatic mutations, and often manifest in early childhood. Hereditary Hemorrhagic Telangiectasia (HHT) is a VA characterized by arteriovenous malformations (AVMs) and dilated capillaries that are visible as red markings on the skin (telangiectasia). Patients suffer from recurrent bleeding (Hemorrhages) with increased incidence of anemia, heart failure and stroke. 80-90% of HHT cases are caused by loss-of-function (LOF) mutations in ACVRL1 (ALK1) or ENG (endoglin). Also, gain-of-function (GOF) mutations are known to cause VAs, like in the case of TIE21914F causing venous malformations (VMs). In spite of many known causative mutations, our understanding of disease mechanisms is incomplete, and therapies are limited. Therapeutic modalities, such as cell-penetrating and tissue-homing peptides, specific towards the cardiovascular system are highly promising as they open the potential to targeted therapies.

In this thesis we generated induced pluripotent stem cell (iPSC)-derived endothelial cell (EC) models for HHT1 (causative gene ENG), HHT2 (causative gene ALK1) and VMs by knocking out ENG or ALK1 or creating a point mutation in TIE2, respectively. In paper I, HHT models were utilized for drug screening to find further medicines which can be repurposed for HHT therapies. Unlike previous cell models where the Tie2-mutant protein was overexpressed, in paper II we applied a mutation knock in strategy that generated endogenous expression levels, thereby resembling the patient situation and examined TIE2 pathway signaling in this model.

In paper III, we identified cell-penetrating peptides (CPPs) in in vivo phage display and explored their targeted delivery capabilities. Using the CPPs we could demonstrate uptake of small interfering RNA (siRNA)-CPP-conjugates specifically into ECs.

In paper IV, we utilized the phage display platform and identified heart-targeting peptides in a myocardial ischemia reperfusion injury (IRI) mouse model that specifically bind cardiomyocytes in the myocardial infarction region. We demonstrated binding of peptides to healthy cardiomyocytes, and furthermore two peptides binding to hypoxic and ischemic cardiomyocytes.

List of scientific papers

I. Kohl F, Roscales M, Keith B, Krimpenfort LT, Firth M, Hong X, Elgendy R, Lazovic B, Queiro Palou A, Hicks R, Jakobsson L, Wiseman J. iPSC- derived ENG and ALK1 KO endothelial cells facilitate modeling of Hereditary Hemorrhagic Telangiectasia and build up a platform for drug screening. [Manuscript]

II. Lazovic B, Nguyen H-T, Ansarizadeh M, Wigge L, Kohl E, Li S, Carracedo M, Kettunen J, Krimpenfort LT, Elgendy R, Richter K,-De Silva L, Bilican B, Singh P, Saxena P, Jakobsson L, Hong X, Eklund L, Hicks R. Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2^L914F mutation in endothelial cells. Angiogenesis. 2024, 27, 523-542. https://doi.org/10.1007/s10456-024-09925-9

III. Kohl F, Laufkötter O, Firth M, Krimpenfort LT, Mangla P, Ansarizadeh M, Geylan G, Eklund L, De Maria L, Jakobsson L, Wiseman J. Identification of cell type-specific cell-penetrating peptides through in vivo phage display leveraged by next generation sequencing. Biomed. Pharmacother. 2025, 182, 117740. https://doi.org/10.1016/j.biopha.2024.117740

IV. Ivanova A, Kohl F, González-King Garibotti H, Chalupska R, Cvjetkovic A, Firth M, Jennbacken K, Martinsson S, Silva AM, Viken I, Wang Q-D, Wiseman J, Dekker N. In vivo phage display identifies novel peptides for cardiac targeting. Sci. Rep. 2024, 14, 12177. https://doi.org/10.1038/s41598-024-62953-9