The development of acid degradable linkers that rapidly hydrolyze at endosomal pHs is a central problem in the field of drug delivery. Acid degradable linkages are challenging to develop because of their instability. In this report we present a new acid degradable linker based on an azide-acetal, which rapidly hydrolyzes at endosomal pHs but has exceptional stability at pH 7.4. The azide-acetal linkage hydrolyzes via a two-step mechanism that requires reduction and acid hydrolysis and has a unique combination of stability and rapid triggerable hydrolysis. The azide-acetal has a hydrolysis half-life of days at pH 7.4 and can be conveniently synthesized and incorporated into delivery vectors, however after in situ reduction with DTT it hydrolyzes with a t1/2<15 minutes at pH 6.0. We used the azide-acetal linker to synthesize acid degradable analogs of the lipid components of lipid nanoparticles and demonstrate that these new lipids are significantly better at delivering mRNA to mice than traditional lipids. Lipid nanoparticles containing acid degradable lipids transfected multiple non-liver organs after an intravenous injection and efficiently transfected brain tissue after an intracranial injection. The azide-acetal linkage has the potential to solve the instability problems associated with acid degradable linkers and has numerous applications in drug delivery.

Gene therapy has experienced an increasing number of successful human clinical trials, leading to 6 FDA approved products using delivery vectors based on adeno-associated viruses (AAV). These successes were possible due to the identification of specific disease targets for which natural variants of AAV were sufficient. However, vectors face a number of barriers and shortcomings that preclude their extension to most human diseases, including limited delivery efficiency to target cells, pre-existing antibodies against AAVs, suboptimal biodistribution, limited spread within tissues, and/or an inability to target delivery to specific cells. These barriers are not surprising, since the parent viruses upon which vectors are based were not evolved by nature for our convenience to use as human therapeutics. Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements.

As an alternative, for over two decades we have been implementing directed evolution – the iterative genetic diversification of the viral genome and functional selection for desired properties – to engineer highly optimized, next generation AAV variants for efficient and targeted delivery to any cell or tissue target. We have genetically diversified AAV using a broad range of approaches from fully random (e.g. error prone PCR) to computationally guided (e.g. by machine learning). The resulting large (~109) libraries are then functionally selected for substantially enhanced delivery, yielding AAVs capable of highly efficient therapeutic gene delivery. Our variants have been effective in both animal models and in 6 human clinical trials to date, and results from both will be discussed.