Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno-associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the-art rAAV formulations and can further serve as a map for rational formulation development activities in the future.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by ¹¹¹In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

Recombinant adeno-associated viral vectors (rAAV) are currently considered the most promising platform for in vivo gene therapy. Several products have already achieved market approval, and many more are in the clinical pipelines, targeting a wide range of tissues and diseases. As we have recently reviewed, most rAAV products are still delivered in a deep-frozen state. However, efforts are emerging to enable storage of gene therapy products at higher temperatures. The main focus of these efforts can be grouped into vector optimization and formulation development. To efficiently screen formulation compositions, several laboratory-scale unit operations need to be taken into account. These operations require freeze–thaw cycles, handling at room temperature, and filtration steps introducing interfacial stress, all of which can potentially impact the quality attributes of rAAV vectors. In this article, we examine a number of critical unit operations that need to be conducted in order to screen rAAV formulations. These include freeze–thaw cycles, buffer exchange, sterile filtration, and in-use compatibility of clinically relevant rAAV vectors carrying a reporter gene. Regarding thermal stability, our focus is on short- to mid-term stability, which is applicable to the handling of rAAV samples in the laboratory, rather than on conditions for long-term storage. We included rAAV2, one of the most sensitive serotypes, as a ‘worst-case scenario’ for most stresses, as well as rAAV9 and engineered vectors. Our data show that formulations with neutral pH, high ionic strength, and the addition of sucrose have increased stability against thermal stress and multiple freeze–thaw cycles. Formulations at slightly acidic pH (pH 5.2) are beneficial for physico-chemical stability but have a detrimental effect on potency. Upon shaking, none of the commonly used surfactants (poloxamer-188, polysorbate-20, or -80) prevent aggregation. Therefore, preventing adsorption of the capsids to the contact materials remains the main purpose of including a surfactant in rAAV formulations. Buffer exchange can introduce significant stress to rAAV vectors in a serotype-dependent manner. Dialysis was shown to be the safest method to prevent titer loss and aggregation during buffer exchange, due to the minimal shear stress it introduces. Lastly, we found that sterile filtration does not significantly impact the quality attributes of rAAVs, taking rAAV8-based capsids as a representative example. In conclusion, we believe that the findings presented and the overall experience we share in this work will support the handling of rAAV vectors during research and development activities. Graphical Abstract