Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Postnatal establishment of allelic Gαs silencing as a plausible explanation for delayed onset of parathyroid hormone resistance owing to heterozygous Gαs disruption.

Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of α-subunit of the stimulatory G protein (Gαs) activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH resistance caused by the maternal loss of Gαs, ie, hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (3 weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH resistance in patients with PHP-Ia.

Impaired adaptation to repeated restraint and decreased response to cold in urocortin 1 knockout mice.

Urocortin 1 (UCN1) is a corticotropin-releasing factor (CRF)-like peptide whose role in stress is not well characterized. To study the physiological role of UCN1 in the response of the hypothalamic-pituitary-adrenal (HPA) axis to stress, we generated UCN1-knockout (KO) mice and examined their adaptation to repeated restraint and to cold environment. Wild-type (WT) and UCN1-KO animals were restrained hourly for 15 min from 9 AM to 2 PM, and blood samples were obtained for corticosterone measurement. WT animals adapted to repeated restraint with a decreased corticosterone response; the restraint-stimulated corticosterone levels fell from 215 +/- 31 ng/ml in naïve animals to 142 +/- 50 ng/ml in mice subjected to repeated restraint (P < 0.01) and from 552 +/- 98 to 314 +/- 58 ng/ml (P < 0.001) in males and females, respectively. Male UCN1-KO mice did not show any adaptation to repeated restraint; instead, restraint-stimulated corticosterone levels were increased from 274 +/- 80 ng/ml in naïve animals to 480 +/- 75 ng/ml in mice subjected to repeated restraint (P < 0.001). Female UCN1-KO mice showed only a partial adaptation to repeated restraint, with a decrease in the restraint-stimulated corticosterone response from 631 +/- 102 ng/ml in naïve animals to 467 +/- 78 ng/ml in mice subjected to repeated restraint (P < 0.01). In addition, UCN1-KO mice showed no corticosterone response to 2-h cold environment. These data demonstrate an important role for UCN1 in the HPA axis adaptation to repeated restraint and in the corticosterone response to a cold environment.

Impact of impaired receptor internalization on calcium homeostasis in knock-in mice expressing a phosphorylation-deficient parathyroid hormone (PTH)/PTH-related peptide receptor.

Internalization of G protein-coupled receptors (GPCRs) and desensitization of the hormonal responses are well characterized in vitro for several hormonal systems. The physiological role of internalization for a GPCR receptor involved in homeostatic functions has not been established, although it has been assumed based on in vitro data. We have previously shown that phosphorylation of the PTH/PTHrP receptor is required for its internalization and for the desensitization of the responsiveness to PTH and PTHrP in vitro; the internalization and desensitization response is impaired in a PTH/PTHrP receptor mutant bearing serine to alanine mutations in the phosphate acceptor sites. To understand the physiological role of receptor internalization on calcium homeostasis, we have knocked-in the internalization-impaired PTH/PTHrP receptor mutant using homologous recombination technology. The genetically modified animals exhibited calcium levels no different from control animals, but PTH levels were one third of those in control animals indicating that homeostasis could be maintained only by 3-fold suppression of PTH secretion. We also analyzed the calcemic response to PTH in vivo. Here we show that mice expressing the internalization-impaired PTH/PTHrP receptor mutant have dramatically exaggerated cAMP and calcemic responses to sc PTH administration when compared with control animals given the same dose. These data show for the first time the role of G protein receptor phosphorylation and internalization per se in the regulatory function of an endocrine system controlled by a GPCR.