effective-safe-gene-delivery-to-mouse-kidney-via-slow-retrograde

Effective and Safe Gene Delivery to the Mouse Kidney <em>via</em> Slow Retrograde Renal Pelvis Injection of Adeno-Associated Virus Vectors

The development of effective in vivo tools and methods for gene delivery to the kidney is crucial for advancing basic kidney research and gene therapy for ...

Oregon Health & Science University

The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity. To demonstrate proof-of-principle for this approach a transgenic mouse was generated harbouring a knock-in of a scgRNA into the Cytokeratin 14 (Krt14) locus. Krt14 expression marks the stem cells of squamous epithelium in the skin and oral mucosa. The scgRNA targets a Stop cassette preceding a fluorescent reporter in the Ai9-tdtomato mouse. Ai9-tdtomato reporter mice harbouring this allele along with a spCas9 transgene demonstrated precise marking of the Krt14 lineage. We conclude that RNA polymerase II promoter driven scgRNAs enable the use of CRISPR/spCas9 for genetic lineage tracing.

In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9.

The G protein-coupled receptor 108 () gene encodes a protein factor identified as critical for adeno-associated virus (AAV) entry into mammalian cells, but whether it is universally involved in AAV transduction is unknown. Remarkably, we have discovered that  is absent in the genomes of birds and in most other sauropsids, providing a likely explanation for the overall lower AAV transduction efficacy of common AAV serotypes in birds compared to mammals. Importantly, transgenic expression of human  and manipulation of related glycan binding sites in the viral capsid significantly boost AAV transduction in zebra finch cells. These findings contribute to a more in depth understanding of the mechanisms and evolution of AAV transduction, with potential implications for the design of efficient tools for gene manipulation in experimental animal models, and a range of gene therapy applications in humans.

Genomic loss of GPR108 disrupts AAV transduction in birds.

AAV vectors show promise for gene therapy; however, kidney gene transfer remains challenging. Here we conduct a barcode-seq-based comparison of 47 AAV capsids administered through different routes in mice, followed by individual validation. We find that local delivery of AAV-KP1, but not AAV9, via the renal vein or pelvis effectively transduces proximal tubules with minimal off-target liver transduction, while systemic AAV9, but not AAV-KP1, enhances proximal tubule and podocyte transduction in chronic kidney disease. We demonstrate that these contrasting observations are partly due to differences in their pharmacokinetics. Importantly, we show that renal pelvis injection overcomes pre-existing immunity, leading to robust and exclusive proximal tubule transduction, in non-human primates (NHPs). In addition, we highlight drastic differences in renal transduction profiles between mice and NHPs. Thus, this study provides mechanistic insights and underscores importance of context-dependent selection of AAV capsids to overcome challenges in gene delivery to the kidney.

Article	Year
In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9. Developmental biology\| PubMed ID: 37813160	2023
Genomic loss of GPR108 disrupts AAV transduction in birds. bioRxiv : the preprint server for biology\| PubMed ID: 38798475	2024
Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids. Nature communications\| PubMed ID: 39737896	2024

Anusha Sairavi

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