桿狀病毒改質間葉幹細胞的臨床前安全性評估及利用桿狀病毒調控MicroRNA Sponges修復骨質疏鬆骨缺陷模型

Preclinical Safety Evaluation of Baculovirus-Engineered Mesenchymal Stem Cells and Healing of Osteoporotic Bone Defects Using Baculovirus-mediated MicroRNA Sponges

李奎璋(Li, Kuei Chang)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/博士(2015年)

摘要

我們最近開發了新型的混成桿狀病毒並且可以有效的轉導間葉幹細胞。此新型混成桿狀病毒結合FLPo/Frt系統，可以延長轉殖基因表現並應用在兔子的軟骨及硬骨修復上。為了將此技術推向臨床應用，本研究第一部分評估此新型桿狀病毒轉導人類脂肪間葉幹細胞體外安全性及植入桿狀病毒轉導豬脂肪間葉幹細胞的免疫安全性。我們發現混成桿狀病毒轉導，不會影響人類脂肪間葉幹細胞的存活率、免疫抑制能力和幹細胞特定的表面抗原表現。此外混成桿狀病毒不會造成轉導細胞染色體的變異，外源基因也不會嵌入細胞染色體中，也不會在小鼠上造成腫瘤。在免疫安全性評估中，我們以會表現人類骨形態蛋白第二型(BMP2)和血管內皮細胞生長因子(VEGF)的混成桿狀病毒轉導豬脂肪間葉幹細胞，再植入迷你豬股骨缺陷處。我們發現混成桿狀病毒轉導豬脂肪間葉幹細胞可以延長BMP2/VEGF的表現並且有效的修復大範圍股缺陷，但僅刺激短暫的抗BMP2/VEGF抗體、細胞激素的表現及移植處免疫細胞的浸潤。上述結果證明新型的混成桿狀病毒可以安全地應用於間葉幹細胞改質上和骨組織工程的應用上，因此具有臨床應用的潛力。
本研究第二部分是利用桿狀病毒結合Cre/loxP系統延長表現MicroRNAs sponge進行骨質疏鬆模型的骨修復。MicroRNAs是一群內生性未編碼 RNA並且參與許多基因調控，包含蝕骨細胞成熟、造骨細胞分化及骨生成。最近有許多研究顯示在骨質疏鬆症患者存有異常表現量的miRNAs，雖然目前有許多藥物治療骨質疏鬆症，但卻很少針對骨質疏鬆症造成骨缺陷的治療方針，因此我們嘗試利用miRNAs進行骨質疏鬆的骨修復。我們發現在骨質疏鬆的大鼠骨髓間葉幹細胞中有過量表現的microRNAs (miR-30b, miR-138, miR-140和miR-214)。桿狀病毒調控的miR-140和miR-214 sponge所轉導之骨質疏鬆的骨髓間葉幹細胞具有增加骨分化能力與減緩蝕骨細胞成熟能力。接著我們植入受BMP2/miR-214 sponges桿狀病毒轉導的骨質疏鬆的骨髓幹細胞到股骨缺陷的骨質疏鬆大鼠中(LEBW/214S組)，其結果可以發現和non-operated OVX組(骨修復率22%)相比下，LEBW/214S組在術後第4週骨修復率達到28%。LEBW/214S組不但加速骨修復而且改善新生骨組織的品質(增加骨密度、平均海綿骨厚度、海綿骨數量及降低平均海綿骨間距)，此研究提供一個新穎的治療方式應用在骨質疏鬆的骨修復上。
總結來說，我們證實了FLPo/Frt混成桿狀病毒載體系統可以安全地應用於脂肪間葉幹細胞改質上與大型動物的骨修復。此外，我們也證實了Cre/loxP混成桿狀病毒所調控的骨質疏鬆的骨髓間葉幹細胞(表現BMP2/miR-214 sponges)可以修復骨質疏鬆的骨缺陷。

關鍵字

組織工程 ； 幹細胞 ； 桿狀病毒 ； 臨床安全性評估 ； microRNA ； 骨質疏鬆

並列摘要

We recently developed hybrid baculovirus (BV) vectors that exploited FLPo/Frt-mediated DNA minicircle formation. Engineering of adipose-derived stem cells (ASCs) with the FLPo/Frt-based BV vectors enabled prolonged transgene expression and, after cell implantation into rabbits, ameliorated cartilage regeneration and bone repair. To translate the hybrid BV one step further towards clinical applications, here we assessed the biosafety profiles of the hybrid BV-engineered human ASCs (hASCs) in vitro and evaluated the immune responses elicited by the engineered porcine ASCs (pASCs) in large animals. We confirmed that the hybrid BV did not compromise the hASCs viability, immunosuppressive capacity and surface characteristics. Neither did the hybrid BV cause chromosomal abnormality/transgene integration in vitro nor induce tumorigenicity in vivo. In the large animal study, pASCs were engineered with the hybrid BV expressing BMP2/VEGF and implanted into femoral bone defects in mini pigs. The hybrid BV-engineered pASCs enabled prolonged BMP2/VEGF expression and triggered the healing of massive segmental bone defects, while only eliciting transient antibody, cytokine and local cellular immune responses stemming from the implantation procedure itself. These data altogether demonstrated the safety of the hybrid BV vectors for ASCs engineering and bone healing in large animals, hence implicating the potential in clinical applications.
In the second part of study, we developed new BV vectors that exploit Cre/loxP-mediated microRNAs (miRNAs) sponge expression to treat osteoporotic bone defects. MicroRNAs (miRNAs) are a class of small non-coding, single-stranded RNAs, which are important regulators of various biological processes, including osteoclastogenesis, osteoblast differentiation and bone formation. Recently, many studies revealed aberrant miRNAs expression in elderly osteoporotic patients. Although many pharmacological agents prevent osteoporotic fractures, the repair of bone defects following fracture draws much less attention. Therefore, we have attempted to utilize miRNAs for repairing osteoporotic bone defects. Here, we found that bone marrow mesenchymal stem cells (BMSCs) harvested from rats with long-term estrogen deficiencies exhibited over-expression of miRNAs level (miR-30b, miR-138, miR-140 and miR-214). We unveiled that the osteogenic differentiation of osteoporotic BMSCs was enhanced by BV-mediated miR-140 or miR-214 sponges transduction, and also mitigated osteoclast maturation via a paracrine fashion after down-regulation of miR-140 or miR-214 in osteoporotic BMSCs by the BV transduction. Allotransplantation of the BMP2/miR-214 sponges-expressing osteoporotic BMSCs (LEBW/214S group) into the critical-size defect (3 mm in diameter) at the femur metaphysis of ovariectomised rat potentiated the bone healing and remodeling, filling 28% of bone volume/total volume (BV/TV) at 4 weeks in comparison to non-operated OVX group (22% of BV/TV). The LEBW/214S group not only accelerated the healing, but also ameliorated the bone quality (increasing density, trabecular number, trabecular thickness and decreasing trabecular space), as evaluated by micro computed tomography, histology and immunohistochemical staining. This study provided a new avenue to treatment of osteoporotic bone defects using miRNA-modulated BMSCs.
Taken together, we demonstrated the safety of the FLPo/Frt hybrid BV vectors for ASCs engineering and bone healing in large animals. Furthermore, we also confirmed the feasibility of the Cre/loxP hybrid BV-mediated BMP2/miR-214 sponges-expressing osteoporotic BMSCs for osteoporotic bone healing.

並列關鍵字

tissue engineering ； stem cells ； baculovirus ； preclinical safety evaluation ； osteoporosis

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