Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids.

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.

Two new members of the Tetrahymena multi-stress-inducible metallothionein family: characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes.

We report the cloning and characterization of two new metallothionein (MT) genes (TrosMTT1 and TrosMTT2), isolated as cDNAs, from the ciliated protozoa Tetrahymena rostrata. The TrosMTT1 inferred protein has been identified as a CdMT and included into the 7a subfamily of Tetrahymena MTs, while TrosMTT2 has been identified as a CuMT (including it into 7b subfamily), due to its similarity to TpigMT-2 and its significant induction by copper. TrosMTT1 protein sequence reveals a remarkably regular and hierarchical modular organization, as it is known for other Tetrahymena CdMTs, showing a bi-modular structure. TrosMTT2 presents a structural organization based on CKCX(2-5)CKC repeats, like it occurs in other Tetrahymena CuMTs, indicating that an evolutionary history based on intra-gene duplications might be also possible. Both are also multi-stress-inducible genes because they are induced by other heavy metals and stressors, as it has been shown by quantitative real-time RT-PCR. It is the first time that the gene expression of a putative Tetrahymena CuMT is analyzed by quantitative PCR, confirming it as a CuMT. These two new Tetrahymena MTs complete, at present, the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs. Furthermore, both, a comparative analysis of relative gene expression values obtained by quantitative RT-PCR on other Tetrahymena MT genes and an analysis of the different Tetrahymena MTs based on the different Cys clusters of these proteins are carried out, which show an update view of Tetrahymena MT gene family.