Oral Delivery of Angiotensin-Converting Enzyme 2 and Angiotensin-(1-7) Bioencapsulated in Plant Cells Attenuates Pulmonary Hypertension

Abstract

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin–angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics.

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

“…Fusion of the (nontoxic) Cholera toxin B subunit (CTB) to a reporter protein (GFP) expressed in chloroplasts and bioencapsulated in plant cells was orally delivered across the gut epithelium through GM1 receptors and furin cleavage site engineered between CTB-GFP released GFP into circulation or different tissues [9]. Fusion of CTB to therapeutic proteins facilitate their effective oral delivery for induction of oral tolerance [10–13] or deliver functional proteins to sera [5,14,15] or across blood brain or retinal barriers [15,16]. …”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Therefore, oral delivery of proinsulin or exendin-4 reduced blood sugar levels similar to injected proteins (Boyhan and Daniell, 2011;Kwon et al, 2013). Oral delivery of angiotensin and ANGIOTENSIN-CONVERTING ENZYME2 expressed in chloroplasts reversed or prevented pulmonary hypertension by shifting the renin-angiotensin system to its protective axis, resulting in a decrease in fibrosis, improvement in cardiopulmonary structure and function, and restoration of right heart function (Shenoy et al, 2014). Furthermore, ocular inflammation caused by decreased activity of the protective axis of the renin-angiotensin system was improved significantly .…”

“…Therefore, transgenes inserted into chloroplast genomes are expressed at high levels, up to 70% of total leaf protein (De Cosa et al, 2001;Ruhlman et al, 2010). A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014). Most importantly, expressed proteins are highly stable when lyophilized plant cells are stored at ambient temperature (Kwon et al, 2013;Lakshmi et al, 2013;Kohli et al, 2014;Jin and Daniell, 2015).…”

“…However, expression of larger human proteins is a major challenge. For example, cholera nontoxic B subunit (CNTB)-fused native human blood-clotting factor VIII heavy chain (FVIII HC;86.4 kD) or ANGIOTENSIN CONVERTING ENZYME2 (92.5 kD) were expressed at very low levels (Shenoy et al, 2014;Sherman et al, 2014). Likewise, the expression of viral vaccine antigens is quite unpredictable, with high, moderate, or extremely low expression levels (Birch-Machin et al, 2004;Lenzi et al, 2008;Waheed et al, 2011aWaheed et al, , 2011bInka Borchers et al, 2012;Hassan et al, 2014).…”

Hypertension Editors’ Picks

“…Two studies were designed to improve the pharmacological management of PAH using novel drug delivery strategies. 15,16 …”