Biology subjects

As, M.

Publications and source records attributed to As, M..

3 recordsLinked to original sources

bioRxiv2024

Retinal organoids mirror CRISPR/Cas9 gene editing efficiency observed in vivo

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include the presence of all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing optimization, particularly for specific genetic disorders. We assessed the potential of retinal organoids for optimizing CRISPR/Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant Retinitis Pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR/Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual AAV systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/630388v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@94478borg.highwire.dtl.DTLVardef@e50970org.highwire.dtl.DTLVardef@ce998corg.highwire.dtl.DTLVardef@1c2bbdf_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOCRetinal organoids can be used to mirror in vivo mouse retina to develop CRISPR therapeutics. Here, Pulman and colleagues show similar ranges of gene editing and delivery dynamics between the organoids and in vivo mouse retina, highlighting the organoids underexplored potential for evaluating gene editing therapies in retinal diseases.

bioengineering↗

bioRxiv2023

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR- Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Successful delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes has been reported in the retinal pigment epithelium in vivo but not into photoreceptors, the main target of retinal dystrophies. Here, we investigate the feasibility of direct RNP delivery to photoreceptors and RPE cells. We show that RNPs composed of Cas9 or adenine- base editor and guide RNA, without addition of any carrier compounds, induce gene editing in retinal cells at variable rates depending on the guide RNA efficiency and on the locus. But Cas9 RNP delivery at high concentrations leads to outer retinal toxicity indicating a need to improve delivery efficiency for future therapeutic use.

bioengineering↗

bioRxiv2023

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicals in vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily. To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model species Hypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species, H. exemplaris, Acutuncus antarcticus and Paramacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, named Tardigrade DNA damage Response protein 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

molecular biology↗