Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Corticotropin-releasing hormone (CRH) is centrally involved in coordinating responses to a variety of stress-associated stimuli. Recent clinical data implicate CRH in the pathophysiology of human affective disorders. To differentiate the CNS pathways involving CRH and CRH receptor 1 (Crhr1) that modulate behavior from those that regulate neuroendocrine function, we generated a conditional knockout mouse line (Crhr1(loxP/loxP)Camk2a-cre) in which Crhr1 function is inactivated postnatally in anterior forebrain and limbic brain structures, but not in the pituitary. This leaves the hypothalamic-pituitary-adrenocortical (HPA) system intact. Crhr1(loxP/loxP)Camk2a-cre mutants showed reduced anxiety, and the basal activity of their HPA system was normal. In contrast to Crhr1 null mutants, conditional mutants were hypersensitive to stress corticotropin and corticosterone levels remained significantly elevated after stress. Our data clearly show that limbic Crhr1 modulates anxiety-related behavior and that this effect is independent of HPA system function. Furthermore, we provide evidence for a new role of limbic Crhr1 in neuroendocrine adaptation to stress.

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.