To date, most neurodegenerative disorders remain difficult to treat since neither traditional pharmaceutical nor surgical interventions have proven effective for these diseases. Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson’s, Alzheimer’s and Huntington’s diseases pose serious risks on the individual’s health and quality of life. Gene therapy is a promising new approach that can be applied to treat the underlying cause of such disorders and also has the potential to be customized to patients’ needs. Pfizer’s rare disease research unit have been studying a potential gene replacement therapy for Friedrich’s ataxia, an inherited neurodegenerative disease that causes impairment of the extremities, cardiac function, and sometimes vision and hearing. In this disorder, reduced expression of the mitochondrial protein frataxin (FXN) prevents sufficient ATP production in nerve cells in the spinal cord and peripheral nervous system. Therefore, gene therapy can be used to express FXN, compensating for the low levels of this protein and treating the root-cause of the illness.
Generally, in vivo delivery of nucleic acids is pivotal to studying genetic causes of the nervous system disorders and developing gene therapies for them. Viral vectors remain the primary means of transducing genes in neurons in vivo, but they are time consuming and complex to prepare, have limitations to gene length and have limited repeat-dosage potential. Prior publications have demonstrated successful non-viral delivery of short interfering RNA (siRNA) by cortical injection of Neuro9™ lipid nanoparticles (LNPs) and subsequent RNA interference-mediated knockdown of gene products in animal models (see related resources). To compensate for low FXN expression, a treatment can deliver messenger RNA (mRNA) that encodes for FXN. However, delivering mRNA is more challenging than siRNA because mRNA is prone to rapid degradation, and the longer length reduces the number of copies that can be encapsulated and makes endosomal escape less efficient.
In this article, Pfizer researchers demonstrated spinal injection of mRNA-LNPs and the expression of exogenous human FXN protein in dorsal root ganglia of rats. The paper, which appeared in the February 2016 edition of Scientific Reports, suggests that mRNA replacement therapy can be used to treat diseases where mutations cause a loss of protein function. To overcome challenges of mRNA delivery, the authors encapsulated codon optimized mRNA encoding human frataxin into LNPs, using the NanoAssemblr™ Benchtop Instrument to produce in vivo scale LNP doses in minutes. Encapsulation in LNPs protects the mRNA from degradation and mediates efficient uptake and endosomal escape even in difficult to transfect cells such as neurons. Following lumbar administration, expression of FXN was detected in dorsal root ganglia near the injection site, but was not found elsewhere in the CNS. The exogenous human FXN was almost completely processed into the mature form, which only takes place in the mitochondrion. This suggests the protein is bioavailable and could be functioning as intended. Additionally, the exogenous protein was found at three-times the abundance of mouse frataxin. These findings represent the first demonstration of exogenous protein expression in dorsal root ganglia following in vivo mRNA delivery, and mark a milestone towards the development of future nucleic acid therapies for neurodegenerative diseases.
In Friedreich’s ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.