Parvoviruses are a diverse family of small, non-enveloped DNA viruses that infect a wide variety of species, tissues and cell types. For over half a century, their intriguing biology and pathophysiology has fueled intensive research aimed at dissecting the underlying viral and cellular mechanisms. Concurrently, their broad host specificity (tropism) has motivated efforts to develop parvoviruses as gene delivery vectors for human cancer or gene therapy applications. While the sum of preclinical and clinical data consistently demonstrates the great potential of these vectors, these findings also illustrate the importance of enhancing and restricting in vivo transgene expression in desired cell types. To this end, major progress has been made especially with vectors based on Adeno-associated virus (AAV), whose capsid is highly amenable to bioengineering, repurposing and expansion of its natural tropism. Here, we provide an overview of the state-of-the-art approaches to create new AAV variants with higher specificity and efficiency of gene transfer in on-target cells. We first review traditional and novel directed evolution approaches, including high-throughput screening of AAV capsid libraries. Next, we discuss programmable receptor-mediated targeting with a focus on two recent technologies that utilize high-affinity binders. Finally, we highlight one of the latest stratagems for rational AAV vector characterization and optimization, namely, machine learning, which promises to facilitate and accelerate the identification of next-generation, safe and precise gene delivery vehicles.

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24–48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.

Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell-type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease.