Modeling Bietti's crystalline dystrophy by employing patients' induced pluripotent stem cells and CRISPR/Cas9 technique

Abstract

Bietti crystalline dystrophy (BCD) is an incurable disease characterized by crystalline deposition and retinal pigment epithelium (RPE) degeneration. Although CYP4V2 gene mutations are reported in BCD, the pathological mechanisms are mostly unknown because of limitations in human RPE cell-sources and animal models. Recent studies demonstrated the advantage of induced pluripotent stem cells (iPSC) for disease modeling. Here we propose to explore pathological mechanisms of BCD using patients’ iPSC for the development of available therapeutic strategies in BCD treatments.

In chapter 3, two BCD patients and one healthy donor were chosen for iPSC reprogramming. The systematical characterizations indicated that these iPSCs were fully reprogrammed into pluripotency.

In chapter 4, isogenic controls were generated in human pluripotent stem cells (PSC), IMR90 and H9, using CRISPR/Cas9 technique. Accumulated lipids including mono-unsaturated fatty acids (FA) were found in CYP4V2 mutant (CYP4V2mt) PSC, but poly-unsaturated fatty acid (PUFA) was undetectable. Nonetheless, their morphology and pluripotency were not affected.

In chapter 5, human RPE cells were differentiated. Biological characterizations indicated that they were functional and mature RPE cells for further studies.

In chapter 6, compared with the wild-type counterparts, the weaker PUFA hydroxylation and lipid accumulations were observed in polarized CYP4V2mt RPE cells. Genes involved in FA metabolism were notably down-regulated, in agreement with reduced FA uptakes. We concluded that FA metabolism was dysfunctional and excessive FA deposited in CYP4V2mt RPE cells.

In chapter 7, we observed that FA accumulation increased mitochondrial oxidant stresses. This lipotoxicity damaged mitochondria and triggered TP53-independent apoptosis in CYP4V2mt RPE cells. Interestingly, no obvious lipid accumulation and mitochondrial oxidant stress were found in subcultures of CYP4V2mt RPE cells before cell polarizations. We concluded that FA accumulation damaged mitochondria and triggered cell apoptosis in CYP4V2mt RPE cells.

In chapter 8, FA profiles revealed increased PUFA in CYP4V2mt RPE cells. FA accumulations, mitochondrial oxidant stresses, and chronic cell deaths were increased in wild-type RPE cells treated with PUFA at low concentrations. Besides, severely acute cell death was evidenced in CYP4V2mt RPE cells after PUFA treatments. We concluded that PUFA mainly contributed to the FA accumulation-induced lipotoxicity in CYP4V2mt RPE cells, suggesting critical roles of PUFA in the RPE degeneration of BCD.

In chapter 9, an AAV2-CYP4V2 vector was generated to exploit the feasible strategy for BCD treatment. After the gene delivery, impaired functions of CYP4V2mt RPE cells were largely rescued evidenced by FA accumulations, mitochondrial oxidant stresses, impaired mitochondrial respiration, and chronic cell deaths. This strategy may present an effective therapeutic tool to rescue the CYP4V2 mutation-induced RPE cell death.

In summary, by employing iPSC and CRISPR techniques, it reveals that aberrant FA metabolisms are primarily associated with the CYP4V2 mutations-provoked RPE degenerations. More specifically, PUFAs are major FA toxicants to induce CYP4V2mt RPE cell death trough TP53-independent apoptosis pathway. Importantly, the AAV2-CYP4V2 mediated gene therapy effectively recovers impaired CYP4V2mt RPE cell functions. It deserves further verifications in vivo for the development of gene therapy.