首页 > 基因编辑 >  《Nature》最新重磅:CRISPR-Cas9技术取得新突破,发现肿瘤免疫治疗新靶点


2017-07-21  来源:艾兰博曼医学网  作者:鱼会飞  编辑:陌莉花开
丹娜法伯 波士顿儿童癌症和血液病中心开发了一种新型的肿瘤治疗靶向的筛选方法,利用CRISPR-Cas9基因编辑技术对小鼠中数千种肿瘤基因的功能进行检测。研究发现,肿瘤细胞中Ptpn2基因的缺失


儿科肿瘤学家W. Nicholas Haining(BM,BCh)是这个项目的主导者,他表示,这随即将引发一系列的研究,即为其他药物与PD-1抑制剂联用给患者带来的治疗反应。迄今,癌症治疗最大的挑战是找到有效的免疫治疗靶标,并寻找与PD-1抑制剂联合使用的最有效的靶点。所以,我们需要更好的一个系统来确定可能有助于自身免疫系统攻击肿瘤细胞的药物新靶点。有一系列的生物学途径可用于免疫治疗的成功,有很多途径是我们先前不可想象的。同时,没有这种筛选方法,可以增强PD-1免疫治疗效果的Ptpn2是癌症免疫治疗的靶点也是不容易被发现的。

W. Nicholas Haining


Robert Manguso是Haining实验室的在读研究生,他设计了一个基因筛选系统,用于识别帮助癌细胞逃避免疫攻击的基因。使用CRISPR-Cas9技术,系统地敲除黑色素瘤皮肤癌细胞中的2368个基因。然后对哪些基因缺失时癌细胞更容易受到PD-1阻断进行了识别。




随着新的筛选方法的进行,Haining正在扩大自己的筛选范围,从一次筛选数千个基因,到最后能够覆盖到整个基因组,并将扩大到黑色素瘤、肠癌、肺癌、肾癌等其他癌种中。他组建了一个由Dana-Farber/Boston Children和Broad Institute组成的大型科研团队,以应对如此大规模的筛选工作中所带来的技术挑战。虽然更多的新的潜在的药物靶点还在挖掘中,Haining团队已经对Ptpn2展开了进一步的研究。他们正在研究Ptpn2的结构并着手研制一种能够关闭Ptpn2的分子靶向药物。


Novel CRISPR-Cas9 genetic screening approach enables discovery of new drug targets to aid cancer immunotherapy

A novel screening method developed by a team at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center — using CRISPR-Cas9 genome editing technology to test the function of thousands of tumor genes in mice — has revealed new drug targets that could potentially enhance the effectiveness of PD-1 checkpoint inhibitors, a promising new class of cancer immunotherapy.

In findings published online today by Nature, the Dana-Farber/Boston Children’s team — led by pediatric oncologist W. Nicholas Haining, BM, BCh — reports that deletion of the Ptpn2 gene in tumor cells made them more susceptible to PD-1 checkpoint inhibitors. PD-1 blockade is a drug that “releases the brakes” on immune cells, enabling them to locate and destroy cancer cells.

“PD-1 checkpoint inhibitors have transformed the treatment of many cancers,” says Dr. Haining, senior author on the new paper, who is also associate professor of pediatrics at Harvard Medical School and associate member of the Broad Institute of MIT and Harvard. “Yet despite the clinical success of this new class of cancer immunotherapy, the majority of patients don’t reap a clinical benefit from PD-1 blockade.”

That, Haining says, has triggered a rush of additional trials to investigate whether other drugs, when used in combination with PD-1 inhibitors, can increase the number of patients whose cancer responds to the treatment.

“The challenge so far has been finding the most effective immunotherapy targets and prioritizing those that work best when combined with PD-1 inhibitors,” Haining says. “So, we set out to develop a better system for identifying new drug targets that might aid the body’s own immune system in its attack against cancer.

“Our work suggests that there’s a wide array of biological pathways that could be targeted to make immunotherapy more successful,” Haining continues. “Many of these are surprising pathways that we couldn’t have predicted. For instance, without this screening approach, it wouldn’t have been obvious that Ptpn2 is a good drug target for the immunotherapy of cancer.”

Sifting through thousands of potential targets

To cast a wide net, the paper’s first author Robert Manguso, a graduate student in Haining’s lab, designed a genetic screening system to identify genes used by cancer cells to evade immune attack. He used CRISPR-Cas9, a genome editing technology that works like a pair of molecular scissors to cleave DNA at precise locations in the genetic code, to systematically knock out 2,368 genes expressed by melanoma skin cancer cells. Manguso was then able to identify which genes, when deleted, made the cancer cells more susceptible to PD-1 blockade.

Manguso started by engineering the melanoma skin cancer cells so that they all contained Cas9, the “cutting” enzyme that is part of the CRISPR editing system. Then, using a virus as a delivery vehicle, he programmed each cell with a different “single guide RNA” sequence of genetic code. In combination with the Cas9 enzyme, the sgRNA codes — about 20 amino acids in length — enabled 2,368 different genes to be eliminated.

By injecting the tumor cells into mice and treating them with PD-1 checkpoint inhibitors, Manguso was then able to tally up which modified tumor cells survived. Those that perished had been sensitized to PD-1 blockade as a result of their missing gene.

Using this approach, Manguso and Haining first confirmed the role of two genes already known to be immune “evaders” — PD-L1 and CD47, drug inhibitors that are already in clinical trials. They then discovered a variety of new immune evaders that, if inhibited therapeutically, could enhance PD-1 cancer immunotherapy. One such newly-found gene of particular interest is Ptpn2.

“Ptpn2 usually puts the brakes on the immune signaling pathways that would otherwise smother cancer cells,” Haining says. “Deleting Ptpn2 ramps up those immune signaling pathways, making tumor cells grow slower and die more easily under immune attack.”

Gaining more ground

With the new screening approach in hand, Haining’s team is quickly scaling up their efforts to search for additional novel drug targets that could boost immunotherapy.

Haining says the team is expanding their approach to move from screening thousands of genes at a time to eventually be able to screen the whole genome, and to move beyond melanoma to colon, lung, renal carcinoma and more. He’s assembled a large team of scientists spanning Dana-Farber/Boston Children’s and the Broad Institute to tackle the technical challenges that accompany screening efforts on such a large scale.

In the meantime, while more new potential drug targets are likely around the corner, Haining’s team is taking action based on their findings about Ptpn2.

“We’re thinking hard about what a Ptpn2 inhibitor would look like,” says Haining. “It’s easy to imagine making a small molecule drug that turns off Ptpn2.”

In addition to Haining and Manguso, co-authors of the paper are Hans W. Pope, Margaret D. Zimmer, Flavian D. Brown, Kathleen B. Yates, Brian C. Miller, Natalie B. Collins, Kevin Bi, Martin W. LaFleur, Vikram R. Juneja, Sarah A. Weiss, Jennifer Lo, David E. Fisher, Diana Miao, Eliezer Van Allen, David E. Root, Arlene H. Sharpe and John G. Doench.





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