Gene therapy has the potential to treat a wide variety of diseases. Delivering nucleic acids, such as DNA and mRNA, allows for the production of an aberrant or absent protein that is causing the disease. Delivery of genes via viruses is very efficient but falls short because of other issues. Nonviral delivery, on the other hand, struggles with efficiency but has advantages in terms of lack of immunogenicity, ease in production, and carrying capacity. DNA is much more stable than mRNA, and the protein production from DNA persists for a longer time. However, DNA delivered to cells must pass through the nuclear envelope to produce protein. Nuclear penetration with nonviral DNA delivery in vivo has not yet been accomplished. mRNA only needs to be delivered to the cytoplasm. Recent interest in nonviral delivery of mRNA has surged upward because delivery of mRNA to various cells in vivo has proven successful.
Yet mRNA still struggles with nuclease stability, which is a major impediment toward efficient expression. A polyacridine PEG-peptide (PEG-peptide) has been previously used to stabilize DNA against nuclease hydrolysis by binding through ionic and intercalative interactions. Binding of PEG-peptide to DNA results in a PEGylated nanoparticle, or polyplex, and which protects the DNA. The same PEG-peptide was applied to mRNA. To increase the ability of PEG-peptide to bind through intercalation, a reverse complementary strand was hybridized to the mRNA, forming double stranded mRNA (dsmRNA). In a similar manner to DNA, complexing dsmRNA or single stranded mRNA (ssmRNA) with PEG-peptide resulted in formation of PEG-peptide polyplexes. A dsmRNA polyplex was much more resistant to ribonuclease challenge in vitro than a ssmRNA polyplex. The mRNA constructs were tested in vivo by hydrodynamic dosing. dsmRNA was found to be translationally competent by producing a high level of luciferase reporter enzyme in the liver of mice. When the reverse strand length was modified such that it hybridized with only the coding region, leaving the untranslated regions (UTRs) and poly(A) tail single stranded, the in vivo translatability (level of expression) and persistence (duration of expression) of dsmRNA was equivalent to that of ssmRNA. Full hybridization of the reverse strand with the coding region, the UTRs, and poly(A) tail resulted in a decrease of in vivo translatability. However, the circulatory stability (an in vivo measure of resistance to degradation in blood) was greatly increased when the reverse strand was fully hybridized.
The persistence of expression of exogenously delivered mRNA is poor in comparison to DNA. The first step in mRNA decay in the cytoplasm is predominantly poly(A) tail shortening, or deadenylation. To address the persistence issue, mRNA with nonadenosine extensions at the 3’ end of the poly(A) tail was synthesized to inhibit deadenylation-dependent mRNA decay. However, increase of the length of tail extension resulted in a concomitant overall decrease in translatability and no increase in persistence. Hybridization of a DNA oligo to the origin of the tail extension activated endogenous RNase H, cleaving the tail extension, exposing the poly(A) tail, and reactivating the mRNA for high level translation, although no increase in persistence was seen with this strategy. A structured tail extension consisting of two human β-globin 3’UTR sequences increased persistence but also decreased overall translatability. Enzymatic poly(A) tailing of this structured tail extension brought back the translatability but simultaneously lost the persistence gain. While this study on poly(A) tail extension mRNA did not produce a highly active mRNA that had increased persistence, its results may be applicable toward other gene therapy applications.
Other efforts to increase the metabolic stability or persistence of mRNA were pursued. Scavenger receptors on resident liver macrophages remove polyplexes from the blood by phagocytosis. Saturation of the scavenger receptors by coadministration of a scavenger receptor inhibitor resulted in increased circulatory stability of dsmRNA. However the scavenger receptor inhibitor was toxic in mice. Another effort to increase the persistence of gene expression in vivo was utilizing an autogene. Autogenes are able to drive the expression of a DNA-based gene outside of the nucleus. In its final form, the autogene did not produce expression.
It is an exciting time to be in the field of mRNA gene therapy. Hopefully the research presented in this thesis will factor in to the knowledge base that can treat and cure human diseases.