The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a solution of pre-formed lipid nanoparticles and mRNA.

The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a suspension of preformed lipid nanoparticles and mRNA.

A method of restoring activity in phenylalanine hydroxylase is provided. The method comprises exposing the phenylalanine hydroxylase to shikimic acid, a functionally equivalent analogue thereof, a pharmaceutically acceptable salt of shikimic acid or analogue thereof, or combinations thereof. A method of screening for allosteric activators for a target enzyme is also provided comprising the steps of: denaturing the target enzyme with a first chaotropic agent to yield denatured enzyme, incubating the denatured enzyme with a candidate compound under denaturing conditions to allow enzyme refolding, and assaying enzyme activity in the presence of enzyme substrate and a candidate compound; and if enzyme activity of the denatured enzyme was restored in step i) by at least about 10% of residual enzyme activity, denaturing the target enzyme with a second chaotropic agent to yield denatured enzyme, incubating the denatured enzyme with the candidate compound under non-denaturing conditions to allow enzyme refolding, and assaying enzyme activity in the presence of enzyme substrate and the candidate compound, wherein an increase in enzyme activity of at least about 10% of residual enzyme activity indicates that the candidate compound is an allosteric activator of the target enzyme.

This invention provides a range of translatable messenger UNA (mUNA) molecules. The mUNA molecules can be translated in vitro and in vivo to provide an active polypeptide or protein, or to provide an immunization agent or vaccine component. The mUNA molecules can be used as an active agent to express an active polypeptide or protein in cells or subjects. Among other things, the mUNA molecules are useful in methods for treating rare diseases.

Provided herein are expression cassettes for expressing a transgene in a liver cell, wherein the transgene encodes a PAH polypeptide. Also provided are methods to treat phenylketonuria (PKU) and/or to reduce levels of phenylalanine in an individual in need thereof. Further provided herein are vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions and kits for expressing a PAH polypeptide in an individual in need thereof.

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids

Provided herein are recombinant adeno-associated virus (rAAV) compositions that can restore phenylalanine hydroxylase (PAH) gene function in cells, and methods for using the same to treat diseases associated with reduction of PAH gene function (e.g., PKU). Also provided are nucleic acids, vectors, packaging systems, and methods for making the adeno-associated virus compositions.

A method of attaching a molecule-of-interest to a microtube, by co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and the second polymeric solution comprises the molecule-of-interest, thereby attaching the molecule-of-interest to the microtube. An electrospun microtube comprising an electrospun shell, an electrospun coat over an internal surface of the shell and a molecule-of-interest attached to the microtube.

A method of attaching a cell or a membrane-coated particle-of-interest to a microtube is provided. The method comprising: co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and wherein the second polymeric solution comprises the cell or the membrane-coated particle-of-interest, thereby attaching the cell or the membrane-coated particle-of-interest to the microtube. Also provided are microtubes with attached, entrapped or encapsulated cells or membrane-coated particles and methods of using same

Provided herein are expression cassettes for expressing a transgene in a liver cell, wherein the transgene encodes a PAH polypeptide. Also provided are methods to treat phenylketonuria (PKU) and/or to reduce levels of phenylalanine in an individual in need thereof. Further provided herein are vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions and kits for expressing a PAH polypeptide in an individual in need thereof.