Biology subjects

Pulman, J.

Publications and source records attributed to Pulman, J..

3 recordsLinked to original sources

bioRxiv2025

AAV-delivered CRISPR-Cas9 elicits persistent retinal immune responses compared with transient responses to RNP

CRISPR-Cas9 is a powerful gene-editing tool with great potential for treating genetic diseases, including inherited retinal disorders. However, its bacterial origin can induce immune responses that may eliminate transduced cells, threatening editing efficiency. A deeper understanding of CRISPR-Cas9 immunogenicity is therefore needed. Previous studies have shown that systemic delivery via Cas9 induces an immune response, but the detailed inflammation and the impact of the vector remain unclear, especially in immune-privileged organs like the eye. In this study, we found that Cas9 delivered to the retina using adeno-associated virus (AAV) induced persistent inflammation, whereas delivery as naked ribonucleoprotein (RNP) complexes resulted in acute inflammation that faded three weeks post-injection. Inflammation was more severe in the rd10 mouse model of inherited retinal degeneration, which exhibits basal inflammation. These findings provide new insights into vector-dependent immune responses to Cas9 in the eye and highlight potential risks associated with its clinical application. TEASERUnderstanding immune reactions to CRISPR-Cas9 and linking these to their delivery methodology increases their safety.

bioengineering↗

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↗