siRNA-based treatment approach to enhance Gs alpha-mediated hormone signaling

Summary
Gsα is a ubiquitous signaling protein necessary for the actions of numerous hormones, neurotransmitters,
and paracrine/autocrine factors. Gsα deficiency causes end-organ resistance to multiple hormones, including
parathyroid hormone (PTH) and thyroid-stimulating hormone (TSH), as well as growth and skeletal
developmental abnormalities, including short stature, heterotopic ossification, and brachydactyly. This disorder,
pseudohypoparathyroidism (PHP), can arise from coding mutations (PHP1A) or abnormal methylation of the
Gsα-coding GNAS gene (PHP1B). No curative therapies exist for patients with these disorders. This proposal
offers a fundamentally new direction toward correcting Gsα signaling and, thereby, rescuing the multi-hormone
resistance caused by Gsα deficiency. In healthy individuals, the paternal Gsα allele is silenced in some hormone-
responsive tissues, including renal proximal tubules and thyroid. Combined with the defective maternal Gsα
allele in PHP1A and PHP1B patients, this tissue-specific paternal Gsα silencing results in Gsα deficiency in these
tissues. Our novel strategy involves targeting this silencing mechanism to regain expression from the paternal
Gsα allele, thus restoring hormone responsiveness. We recently generated unique cell-based models using
human embryonic stem cells (hESCs) to investigate the mechanisms behind the abnormal Gsα expression and
hormone resistance in PHP1B. Our recent results using these PHP1B cell models and previous mouse studies
indicate that a competition-like mechanism operates between the Gsα promoter and the promoter of an upstream
alternative non-coding GNAS transcript, A/B, in a tissue-specific manner. The A/B promoter is maternally
methylated, confining the expression of A/B to the paternal allele. Active paternal A/B transcription silences the
paternal Gsα promoter in cis. In this proposal, we aim to target the A/B promoter by siRNA to achieve
“transcriptional” A/B silencing and, therefore, derepress paternal Gsα expression in tissues where this critical
silencing mechanism ensues. We will thus determine whether A/B promoter-targeting siRNA boosts Gsα
expression levels and rescues hormone resistance caused by GNAS defects. First, we will design and employ
siRNA directed to the A/B promoter (or scrambled siRNA as control) in hESC-derived cells that normally express
Gsα either monoallelically (renal proximal tubule- and thyroid-like cells) or biallelically (chondrocytes). The effects
of the A/B promoter-targeting siRNA will be assessed on allele-specific Gsα expression, Gsα levels, and Gsα
signaling induced by receptor agonists, including PTH, TSH, and PTH-related peptide (PTHrP). Then, we will
test the ability of this strategy to restore Gsα-mediated hormone signaling in PHP1A- and PHP1B-modeling
hESCs differentiated into renal proximal tubule- and thyroid-like cells. Subsequently, we will administer siRNA
targeting the mouse A/B promoter to a PHP1A mouse model to determine if this treatment corrects the
phenotypes resulting from hormone resistance. This highly significant project has substantial translational
promise, particularly in light of the recent approvals of siRNA-based therapeutics for certain diseases.

Grant Number: 1R21HD117132-01A1
NIH Institute/Center: NIH
Principal Investigator: MURAT BASTEPE