Project description:Maddalena et al. showed that the limited DNA transfer capacity (~4.7kb) of adeno associated viral (AAV) vectors can be expanded up to 14kb with triple AAV vectors for the efficient expression of the therapeutic CDH23 (10.1kb) and ALMS1 (12.5kb) genes.
Project description:The human EXT1 gene has been knockdown in HEK293 cells to improve their performance in producing viral vectors such as AAV. This work compares the gene expression for the wildtype and kd_EXT1 cells, in adherent and suspension cultures.
Project description:To investigate if the truncated PE can be dilivered by dual AAV8 vectors for in vivo prime editing. We injected the dual AAV8 into 10-week-old C57BL/6J mice . Livers were isolated 4 weeks after injection and next generation sequencing showed an average of 1.4% and 5.4% precise prime editing with the low and high AAV doses, respectively (Figure 4D ). This demonstrates that PECO-Mini can be efficiently delivered by dual AAVs for in vivo prime editing.
Project description:Zhu et al. report the application of single-cell RNA-sequencing technology for profiling the cell-specific transgene expression and transcriptome dysregulation in mouse liver following intravenous administration of AAV vectors. By profiling 46,500 mouse liver cells, we have identified 3 separate clusters of hepatocytes (hep1, hep2 and hep3), endothelial cells, Kupffer cells and lymphocytes. Assessment of the AAVrh.10mCherry treated liver demonstrated transgene expression in not only hepatocytes, but in all cell types, with significant cell-type-specific expression heterogeneity. Large numbers of cell type-specific genes were up- and down-regulated in response to the AAV vectors. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.
Project description:Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.
Project description:Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, supplementation of ABCA4 (6.8 kb) via REVeRT improves retinal degeneration and function in a mouse model of inherited blindness. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.
Project description:Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV-manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5 producing HEK293 cells using reversed phase nano liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rela-tive label-free quantitation (LFQ) was performed allowing a comparison of transfected vs. un-transfected cells. Gene ontology enrichment and pathway analysis revealed differential expres-sion of proteins involved in fundamental cellular processes such as metabolism, proliferation and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells thus potentially enabling further improvements of gene therapy product manufacturing.
Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).