Clinical Trial
PHASE 2 : ELAFIN (TIPRELESTAT) FOR PULMONARY ARTERIAL HYPERTENSION
A Phase 2 Study of Elafin in PAH - The NIH/NHLBI has awarded Stanford CVI Professors Marlene Rabinovitch and Roham Zamanian a 5-year, $12.7M grant for their proposal for “A Phase 2 Study of Elafin (Tiprelestat) for the Treatment of Pulmonary Arterial Hypertension (PAH)”. The Data Coordinating Center for the trial, led by Dr. Cathie Spino at the SABER unit at the University of Michigan, was funded by a companion application.
PAH is a disease of progressive obliteration of the lung vasculature that results from elastase mediated degradation of elastin, endothelial dysfunction, smooth muscle cell proliferation and chronic peri- and intravascular inflammation. There is an unmet need to find a therapy that is disease modifying in that it addresses these underlying cellular and molecular features of PAH. This proposal tests the safety and efficacy of human recombinant Elafin (tiprelestat) as a treatment for PAH in a randomized placebo-controlled clinical trial across 10 centers. Elafin is ideally suited for this role as it inhibits neutrophil elastase, suppresses cytokine mediated inflammation, and activates BMPR2 receptor signaling resulting in favorable profile of gene expression in hereditary and other forms of PAH. Beyond the primary endpoint of pulmonary vascular resistance (PVR) and numerous secondary endpoints, the study will evaluate the potential for disease modification – a “first-ever” in the field in PAH Clinical Trials. Beyond the clinical study enrolling centers, the study involves collaborations with three major cores: Harvard University for non-invasive pulmonary vascular imaging and blood volume quantification, UCSF for PK/PD modeling & assessments, and Temple University as the investigational pharmacy.
What makes this proposal even more special is that it is based on the life-long work of Dr. Marlene Rabinovitch and her leadership in investigating the role of elastase inhibitors in vascular diseases and their pleotropic impact on beneficial BMPR2 mediated gene regulation, combined with the leadership of Dr. Roham Zamanian in clinical investigation and clinical trials for new and promising PAH therapies.
BASIC AND TRANSLATIONAL RESEARCH PROJECTS
NEUTROPHIL DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION (PAH)
Dr. Taylor, a former postdoctoral Fellow, found that neutrophils from PAH patients when compared to those from healthy donors as controls, show an increase in adhesion, trans-endothelial migration, degranulation, and release of chromatin as extracellular traps (NETosis). Heightened production and release of elastase explained the propensity to NETosis that causes tissue damage. Proteomic analyses identified increased vinculin, explaining increased adhesion and impaired migration. Transcriptomic analyses of PAH vs. control neutrophils revealed a type I interferon response attributed to heightened expression of the endogenous retrovirus HERV-K that can stimulate an increase in vinculin. in fact, the elevation in neutrophil elastase can also be attributed to HERV-K. Dr. Taylor showed that neutrophil extracellular vesicles contain increased elastase activity and HERV-K from PAH patients are sufficient to cause PAH in experimental animals. As part of the Biomarker Core in the Elafin Phase 2 Clinical Trial we will determine if elafin is reducing neutrophil elastase activity in extracellular vesicles and preventing inappropriate NETosis.
MONOCYTE DYSFUNCTION IN PULMONARY ARTERIAL HYPERTENSION (PAH)
In addition to neutrophils, monocytes are also dysfunctional in PAH in conjunction with elevated levels of HERV-K. Our former postdoc, Dr. Rebecca Harper showed that PAH monocytes have reduced BMPR2 and those derived from stem cells of patients with a BMPR2 mutation show increased adhesion and transendothelial migration accounting for their invasion of the lung blood vessels. Dr Mir Adil more recently related the loss of BMPR2 to activation of a kinase that phosphorylates and de-activates a methylase, KAP1 e that is responsible for repressing HERV-K as well as inflammatory genes on the X chromosome in conjunction with the long non-coding RNA, XIST. We are now investigating a relationship between loss of HERV-K methylase activity and high levels of XIST. When there is excess of this non-coding RNA to sequester microRNAs that inhibit other inflammatory genes. Phosphorylation of KAP1 also seems to cause inflammasome upregulation with an increase in cytokines IL6 and IL18.
SINGLE-CELL IMAGING MAPS INFLAMMATORY CELL SUBSETS TO PULMONARY ARTERIAL HYPERTENSION (PAH) VASCULOPATHY
The results of our studies described above and those of others have therefore linked inflammation and altered immunity in the pathogenesis of PAH. Developing a successful therapy to reverse PAH pathology requires a comprehensive knowledge of the immune cell landscape, its microenvironmental complexities, and how communication with the vascular cells leads to the initiation and progression of disease. We therefore embarked on a collaboration with the laboratory of Michael Angelo at Stanford, led by former post-doctoral fellow Selena Ferrian with former lab member Toshie Saito, to map the immune landscape in pulmonary arteries (PAs) in lung tissue and establish spatial relationships that regulate vascular pathology. Dr. Ferrian used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to interrogate the immune landscape in PAs from idiopathic (IPAH) and hereditary (HPAH) PAH patients. Massive immune infiltration in I/HPAH was observed with intramural infiltration linked to PA occlusive changes. The spatial context of CD141+ dendritic cells expressing SAMHD1, TIM-3 and IDO-1 and MPO+ neutrophils within an immune microenvironment was associated with greater SMC mass and occlusion in hereditary PAH associated with a BMPR2 mutation. Monocyte-derived dendritic cells (Mo-DC) were linked to inflamed endothelial cells and proliferating SMCs. Experimental data with Dr. Aiqin Cao, Research Associate, in cultured cells reinforced the causal relationships between mo-DC in mediating SMC proliferation. These studies set the stage for further analyses of the pathways by which mo-DCs induce SMC proliferation and by which the myeloid cells in particular play a key pathogenetic role in PAH. Current studies identified a unique subset of cells enriched in lung vessels from patients with PAH, particularly in those with a BMPR2 mutation. These cells have markers of monocytes and are myofibroblasts. We have shown that smooth muscle cells from patients with a BMPR2 mutation secrete a substance that we are currently isolating that can convert monocytes to myofibroblasts that contribute to the PAH occlusive pathology.
A Program Project Grant (PPG) to Identify Causal Gene Programs for Vascular Disease Using High Throughput CRISPR Genomics
The NIH/NHLBI has awarded an $11.7M Program Project Grant to Dr, Tom Quertermous and his team of investigators. This program leverages the innovative tools the team has developed to systematically study large numbers of candidate disease genes, define how they integrate into gene programs in vascular cells, test how these programs differ across cell types, vascular beds, disease conditions, and biological sex and thus, identify new genes and pathways that influence risk for vascular diseases.
Dr. Rabinovitch directs Project 3 that focuses on pulmonary arterial hypertension (PAH), a condition attributed to known and unknown genetic factors that is also a complication of many medical conditions including congenital heart disease. Her project aims to identify a shared PAH gene program that is disrupted by many different genetic and environmental factors and that can be targeted to effectively treat PAH. Collaborations with Project 1 focusing on endothelial cells in coronary artery and cerebrovascular disease and with Project 3 focusing on smooth muscle cells in atherosclerosis, will determine differences in gene programs in response to the same perturbation in lung, aortic, coronary and brain endothelial and aortic smooth muscle cells explaining different propensities to PAH and systemic vascular disease. These collaborative studies will be important in informing risk and emerging therapies for different conditions affecting the blood vessel wall. The technology involved is in vivo Perturb-seq in which a mouse with CRISPR inhibitory machinery that is activated in endothelial cells receives guide RNAs that suppress over 200 different PAH genes including those with mutations related to PAH. Single cell RNA -seq will then read out the consequence of perturbing a particular gene on the pattern of altered gene expression in lung endothelial cells The expectation is that multiple genes associated with PAH converge on a common pathway of gene dysregulation that in future studies can be therapeutic strategies. We also anticipate discovering as a program why some mutant genes are deleterious in the pulmonary circulation but protective of systemic vascular disease.
TBX4-MEDIATED PULMONARY ARTERIAL HYPERTENSION (PAH)
TBX4 is the second most common genetic cause of pediatric PAH, and while we see both gain-of-function (GOF) or loss-of-function (LOF) mutations in TBX4 in PAH patients, the molecular mechanism leading to disease development is unknown. The hypothesis being tested by postdoctoral fellow Dr. Mauro Lago Docampo is that GOF and LOF TBX4 mutations lead to similar dysregulation of vascular smooth muscle cell (SMC) gene expression and function, and that TBX4 dysregulation in SMC causes endothelial cell (EC) dysfunction, resulting in PAH. In SMC with endogenous TBX4 deleted using CRISPR/Cas9, wild type TBX4 or GOF or LOF tagged constructs will be introduced and proximal proteomics applied to assess changes in the interaction with co-activators and chromatin remodelers. ChIP-Seq and ATAC-Seq will be used to identify alterations in DNA binding related to differential gene expression, determined by RNA-Seq. CRISPR-editing of TBX4 mutations in SMC will determine how gene expression is impacted by the SMC genetic background. To find modulators of penetrance, induced pluripotent stem cells (iPSCs) differentiated to SMC from TBX4 mutation patients and unaffected carriers will be used in -omic and functional SMC characterization and in interaction with EC in co-culture. We have shown that loss of TBX4 leads to a reduction in BMP10 and impaired activation of the BMPR2 receptor in EC. The result in loss of EC repression of SMC proliferation. One of the targets of BMPR2 signaling is the EC membrane protein TMEM100 Administration of TMEM100 rescues the phenotype of SMC proliferation in SMC-EC co-cultures. We have evidence that this is related to TMEM100 regulation of NOTCH signaling and Restoration of the normal EC phenotype necessary to repress SMC proliferation.
BIOFABRICATED VASCULAR TUBES
In collaboration with Mark Skylar-Scott, BASE Scientist and Assistant Professor in Bioengineering, and under the leadership of former postdoctoral fellow Dr Seo Woo Song and current postdoctoral fellow Dr. Jeonghynun Son we have biofabricated vascular tubes lined by EC and surrounded by SMC enmeshed in fibrin that are perfused under laminar shear stress and pulsatile flow. These tubes are being used to test the impact of PAH mutations such as those in BMPR2 or TBX4.
Image shows Vessel-like fibrin tubes that incorporate SMC and are lined by EC will be used to address non-cell autonomous changes in EC gene expression under physiologic flow. ERG and SOX17.
SOX17 and HIGH SHEAR STRESS
A mutation in SOX17, is linked to PAH, particularly in children with a congenital heart defect such as a ventricular septal defect that causes high shear stress in the pulmonary circulation. Dr. Chongyang Zhang, a postdoctoral fellow has addressed the consequences of loss of SOX17 in PA endothelial cells (ECs) subjected to high shear stree and has uncovered an important interaction with the transcription factor ERG. Indeed multiple SOX17 target genes are co-regulated by ERG. One of these target genes is the transmembrane protein TMEM100 that is also regulated by BMPR2 and TBX4. Dr. Zhang has produced a mouse with SOX17 deleted in endogthelial cells that she subjected to an AV shunt to produce high shear stress in the pulmonary circulation. The more severe PAH produced in this mouse appears to be reversed by delivering TMEM100 to lung endothelial cells using a selective AAV2 vector. The goal now is to understand how TMEM100 is reversing PAH and if the rescue mechanism is the same in mice with loss of TBX4 in lung mesenchymal cells.
EXTRACELLULAR VESICLE SIGNALING, BMPR2 and VASCULAR HOMEOSTASIS
The role of exosome (extracellular vesicle, EV) signaling in the maintenance of vascular homeostasis was investigated. These studies led to the identification of BMPR2 as a critical mediator of EV biogenesis during hypoxia, and the observation that aberrant EV biosynthesis and signaling attributed to loss of BMP2 contributes to the pathogenesis of PAH. Endothelial EVs can be engineered to correct a BMPR2 deficiency, and BMPR2 dependent microRNA cargo plays an instrumental role in repressing aberrant smooth muscle cell (SMC) proliferation during hypoxia. These studies show that EVs are an emerging class of pharmacological agents to treat PAH. Future studies will involve developing strategies to optimize the therapeutic index of EVs with improved targeting and reduced immunogenicity.
BMPR2 MUTATION SUBVERTS ENDOTHELIAL DIFFERENTIATION
This project represents a collaboration between the Rabinovitch Lab and that of Drs. Jesse Engreitz and Xiaojie Qiu, BASE Scientists and Assistant Professors in Genetics. It is under the leadership of Research Associate Kamakshi Bichu and Postdoctoral Fellow Dr. Olga Pushkarev. We use induced pluripotent stem cells from three families with a BMPR2 mutation and control subjects. In each family we have iPSCs from PAH patients with a BMPR2 mutation, in which the mutation is uncorrected or corrected and iPSC from a family member with the same mutation but without PAH as well as a gender and age matched control subject. The iPSCs undergo differentiation to arterial endothelial cells with metabolic labeling of RNA transcripts at each stage of differentiation. These studies will help define the altered trajectory of EC differentiation resulting from a BMPR2 mutation as well as modifier genes that confer PAH.
MODIFIER GENES OF THE BMPR2 MUTATION CAUSE PAH
A collaborative study with Dr. Casey Gifford (BASE Scientist and Assistant Professor in Genetics and Pediatrics and Dr. Isabel Blanco (Professor of Medicine, University of Barcelona) with leadership from Kamakshi Bichu (Research Associate) is combining whole genome sequencing and studies in iPSCs from well phenotype families to find gene variants that confer PAH when there is a BMPR2 mutation.
INCREASING OUTPUT FROM THE NORMAL JAG1 ALLELE IN ALAGILLE
This is a joint project with the laboratory of Dr. Jesse Engreitz (BASE Scientist and Assistant Professor of Genetics) and is led by postdoctoral fellow Dr. Mauro Lago Docampo and Research Associates Drs. Aiqin Cao and Lingli Wang(Rabinovitch Lab) with Dr. Michael Montgomer (Engreitz lab). Alagille Syndrome is caused by heterozygous loss-of-function mutations in JAG1 (95% of cases) or its receptor NOTCH2 (2% of cases). The resulting reduction in JAG1-NOTCH2 signaling, particularly in vascular cells, leads to life-threatening complications including biliary atresia, severe hypoplastic central and peripheral pulmonary arteries, extensive proximal and distal pulmonary arterial stenoses, and right-sided congenital heart defects such as Tetralogy of Fallot. Despite repeated corrective surgical procedures in infancy and early childhood, many patients with Alagille Syndrome experience refractory pulmonary artery hypoplasia and stenoses that ultimately lead to right heart failure. This project explores the potential of genome therapy for Alagille Syndrome, by engineering regulatory DNA sequences to turn up the production of the functional, unaffected allele of JAG1. This is an attractive approach as it would be applicable to the 95% of Alagille Syndrome patients that have loss-of-function variants in JAG1 and would be the first proof of concept strategy for other diseases linked to haploinsufficiency. JAG1 is a dose-sensitive gene, where reduction to 50% normal expression levels is sufficient to affect cellular function in vitro and lead to Alagille-like phenotypes in mouse models. Although, it has never been tested whether restoration of JAG1 function in human patient cells would fully restore healthy gene expression programs and cellular phenotypes, this is a promising strategy even if Alagille patients carry additional disease-causing variants that cooperate with JAG1 to affect the penetrance and expressivity of Alagille Syndrome phenotypes. Our preliminary work demonstrated that exposure of endothelial cells to physiological laminar shear stress induces JAG1 and elastin, and that loss of JAG1-NOTCH2 signaling decreases ELN under this condition. Elastin is critical to blood vessel health, and loss of elastin could underlie vascular dysfunction in Alagille Syndrome. We have also observed abnormalities in endothelial cells in iPSC-derived endothelial cells from Alagille patients.
We will characterize the molecular and cellular phenotypes of Alagille versus healthy iPSC-derived endothelial and smooth muscle cells. With expertise developed by Dr. Engreitz and his group, the study team will combine single-cell 10X Multiome with the BPNet and Activity-by-Contact computational models to build a genome-wide nucleotide-resolution map of transcription in these cells, that will be cultured under conditions of static, physiologic, and pathological flow, to identify key regulators of JAG1. We will systematically mutate regulatory DNA sequences in the JAG1 promoter and identify sequences that can increase JAG1 gene expression by ~2-fold. As a test for the potential therapy, we will use CRISPR prime editing to directly correct coding point mutations in iPSCs from Alagille patients and examine the effects of the correction on JAG1 signaling and downstream cellular phenotypes. We will conduct similar experiments introducing regulatory edits to the JAG1 promoter to up-regulate JAG1 expression from the unaffected allele.