A gene therapy approach for long-term normalization of blood pressure in hypertensive mice by ANP-secreting human skin grafts

Abstract

The use of bioengineered human skin as a bioreactor to deliver therapeutic factors has a number of advantages including accessibility that allows manipulation and monitoring of genetically modified cells. We demonstrate a skin gene therapy approach that can regulate blood pressure and treat systemic hypertension by expressing atrial natriuretic peptide (ANP), a hormone able to decrease blood pressure, in bioengineered human skin equivalents (HSE). Additionally, the expression of a selectable marker gene, multidrug resistance (MDR) type 1, is linked to ANP expression on a bicistronic vector and was coexpressed in the human keratinocytes and fibroblasts of the HSE that were grafted onto immunocompromised mice. Topical treatments of grafted HSE with the antimitotic agent colchicine select for keratinocyte progenitors that express both MDR and ANP. Significant plasma levels of human ANP were detected in mice grafted with HSE expressing ANP from either keratinocytes or fibroblasts, and topical selection of grafted HSE resulted in persistent high levels of ANP expression in vivo. Mice with elevated plasma levels of human ANP showed lower renin levels and, correspondingly, had lower systemic blood pressure than controls. Furthermore, mice with HSE grafts expressing human ANP did not develop elevated blood pressure when fed a high-salt diet. These findings illustrate the potential of this human skin gene therapy approach to deliver therapeutic molecules systemically for long-term treatment of diverse diseases.keratinocytes | human engineered skin | hypertension | multi-drug resistance gene | retroviral vectors A lthough skin can be easily accessed for manipulation, a major challenge of using skin gene therapy to treat systemic diseases has been the difficulty of achieving sustained expression of desired therapeutic genes in a high percentage of keratinocytes (Kc) (1-8), in part because few good markers of Kc stem cells exist for isolation and gene targeting (9-13). Previously, we described how topical colchicine treatment of human skin equivalents (HSE) containing Kc engineered to express the multidrug resistance (MDR) gene could select and enrich for MDR-expressing human Kc progenitor cells because only MDRexpressing Kc were able to pump-out the antimitotic inhibitor and proliferate (5). However, the therapeutic utility of this gene therapy approach in treating systemic diseases was unproven.Gene therapy strategies that have been discussed for the treatment of hypertension include overexpression of genes such as atrial natriuretic peptide (ANP) that are relevant in reducing blood pressure (14,15). ANP is a 126-amino acid peptide hormone synthesized mainly by right atrial cardiomyocytes as an inactive precursor (pro-ANP) (Fig. S1). Pro-ANP is secreted and converted into an active ANP peptide of 28 amino acids (α-ANP) by corin receptors located on the cell surface of cardiomyocytes (16, 17) in response to volume expansion and increases in venous pressure. In target tissues such as kidney and endothelial cells, the active...

Reward-based hypertension control by a synthetic brain–dopamine interface

“…To confirm dopamine-based reward-triggered transgene expression in a prototypical therapeutic context, we chose to link the synthetic dopamine-sensor device to expression of the halflife-optimized (30) atrial natriuretic peptide (ANP; pKR147, P CRE -ANP-pA), a powerful vasodilator attenuating high blood pressure (31)(32)(33). After validation of dopamine-triggered ANP expression in vitro (Fig.…”

Atrial natriuretic peptide and regulation of vascular function in hypertension and heart failure

“…As a result, a prolonged ANP overexpression was achieved with a consequent significant decrease of systolic BP in a hypertensive mouse model. Recently, a very innovative approach has been proposed by Therrien et al [106] who demonstrated a long-term normalization of BP in hypertensive mice by implanting ANP-secreting human skin grafts. The human ANP levels in the grafted mice were within the range that has been shown to exert therapeutic effects in patients.…”

The Local Neuropeptide System of Keratinocytes

“…VIP has been shown to affect the proliferative activity of human keratinocytes in vitro [ 67 ]. The findings that topical treatments of grafted bioengineered human skin with the antimitotic agent colchicine select for keratinocyte progenitors that express ANP in mice suggest that basal production of ANP in normal skin is negligible [ 68 ]. With regards to the oral mucosa, liposomal VIP potentiates DNA synthesis in cultured hamster oral keratinocytes, which suggests a possible response to this peptide [ 69 ].…”