SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency.

Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.

Arginase I mRNA therapy - a novel approach to rescue arginase 1 enzyme deficiency.

Arginase I (ARG1) deficiency is an autosomal recessive urea cycle disorder, caused by deficiency of the enzyme Arginase I, resulting in accumulation of arginine in blood. Current Standard of Care (SOC) for ARG1 deficiency in patients or those having detrimental mutations of ARG1 gene is diet control. Despite diet and drug therapy with nitrogen scavengers, ~25% of patients suffer from severe mental deficits and loss of ambulation. 75% of patients whose symptoms can be managed through diet therapy continue to suffer neuro-cognitive deficits. In our research, we demonstrate in vitro and in vivo that administration of ARG1 mRNA increased ARG1 protein expression and specific activity in relevant cell types, including ARG1-deficient patient cell lines, as well as in wild type mice for up to 4 days. These studies demonstrate that ARG1 mRNA treatment led to increased functional protein expression of ARG1 and subsequently an increase in urea. Hence, ARG1 mRNA therapy could be a potential treatment option to develop for patients.

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA.

mRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5'UTR and 3'UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5' UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5'UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.

Leveraging Rational Protein Engineering to Improve mRNA Therapeutics.

Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6-12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro. We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo.

G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a.

Glycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase-/- mouse model (L-G6PC-/-) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.