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Updated: Aug 23, 2026
Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease
Published on: April 19, 2015
Clofarabine Enhances the Transduction Efficiency of Recombinant AAV2 in the Retina
Yuyao Diao1, Xuewei Xiong1, Jiayan Liu1,2
1Department of Ophthalmology, First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, China.
Purpose:
Ribonucleotide reductase inhibitors, a class of antineoplastic agents, were investigated for their ability to enhance recombinant adeno-associated virus serotype 2 (rAAV2) transduction in the mouse retina and their underlying mechanisms.
Methods:
Candidate ribonucleotide reductase inhibitors were screened in ARPE-19 cells to identify concentrations preserving cell viability while optimizing rAAV2 transduction. Clofarabine, which demonstrated superior enhancement of rAAV2 in vitro, was selected for in vivo evaluation via subretinal coadministration with rAAV2.GFP in mice. Transcriptomic mechanisms were dissected using RNA sequencing (RNA-seq) of clofarabine-treated ARPE-19 cells and single-cell RNA-seq of murine retinas after combinatorial treatment.
Results:
Cellular viability assays demonstrated that clofarabine pretreatment significantly enhanced rAAV2.GFP transgene expression in ARPE-19 cells, elevating both mRNA and protein levels compared with rAAV2.GFP transduction alone. This enhancement was mirrored in vivo, where subretinal coadministration of clofarabine with rAAV2.GFP in mice increased retinal transduction efficiency markedly, without detectable toxicity. Transcriptomic profiling delineated clofarabine's mechanism. (1) In proliferating cells, it triggered S-phase arrest by upregulating CCNE2 and CDC6, synchronizing cell populations to optimize viral genome processing; and (2) in postmitotic retinal cells, it suppressed innate immune pathways while enhancing nucleotide biosynthesis and transcriptional activity, thereby creating a microenvironment permissive to rAAV transduction.
Conclusions:
Clofarabine safely enhances rAAV2 transduction efficiency in both ocular cell models and murine retinas. Its ability to synchronize cell cycles in dividing cells and reprogram transcriptional landscapes in postmitotic cells positions it as a promising adjunct for rAAV-based ocular gene therapy, potentially decreasing therapeutic vector doses and improving clinical outcomes.
Insights
Clofarabine, an antineoplastic agent, enhances adeno-associated virus serotype 2 (AAV2) gene therapy in the eye. This drug improves AAV2 transduction in retinal cells by synchronizing cell cycles and optimizing the cellular environment.
Area of Science:
- Ophthalmology
- Gene Therapy
- Molecular Biology
Background:
- Recombinant adeno-associated virus serotype 2 (rAAV2) is a key vector for ocular gene therapy.
- Enhancing rAAV2 transduction efficiency is crucial for improving therapeutic outcomes.
- Antineoplastic agents are being explored for novel applications beyond cancer treatment.
Purpose of the Study:
- To investigate the potential of ribonucleotide reductase inhibitors to enhance rAAV2 transduction in the mouse retina.
- To elucidate the underlying molecular mechanisms by which these inhibitors affect rAAV2 transduction.
- To assess the safety and efficacy of clofarabine as an adjunct for ocular gene therapy.
Main Methods:
- Screening of ribonucleotide reductase inhibitors in ARPE-19 cells to determine optimal concentrations for rAAV2 transduction.
- In vivo evaluation of clofarabine via subretinal coadministration with rAAV2.GFP in mice.
- Transcriptomic analysis using RNA sequencing (RNA-seq) and single-cell RNA-seq to dissect cellular mechanisms.
Main Results:
- Clofarabine significantly enhanced rAAV2.GFP transgene expression in ARPE-19 cells, increasing both mRNA and protein levels.
- In vivo studies showed markedly increased retinal transduction efficiency with clofarabine coadministration, without detectable toxicity.
- Transcriptomic profiling revealed clofarabine induces S-phase arrest in proliferating cells and suppresses innate immunity while enhancing nucleotide biosynthesis in postmitotic retinal cells.
Conclusions:
- Clofarabine safely and effectively enhances rAAV2 transduction efficiency in ocular models.
- Its mechanisms involve cell cycle synchronization and reprogramming of transcriptional landscapes.
- Clofarabine shows promise as an adjunct therapy for rAAV-based ocular gene therapy, potentially reducing vector doses and improving clinical results.
