基因治療用腺病毒載體。使用腺病毒的新基因插入細胞。如果治療是成功的，新的基因，將產生功能性蛋白來治療疾病
把基因表达的工作交给病毒载体[选购宝典]
摘要：
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
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为了将外源的核酸导入细胞，人们常常采用化学转染和电穿孔的方法。随着细胞调控的谜团不断被解开，神经细胞、干细胞、原代细胞以及一些不分裂的真核细胞也成为实验中的宠儿。不幸的是，这些细胞类型却并不配合标准的转染方法。研究人员要么放弃他们的研究，要么接受低转染效率。其实，他们还有一个选择，那就是病毒转导。
据斯克里普斯研究所（Scripps Research Institute）免疫学和微生物学教授John Elder介绍，病毒载体的存在已有数亿年。现代的实验室已经驯服了病毒，确保它既安全又有效，同时利用其能力来转导高比例的细胞。
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
制备病毒
利用病毒作为载体就已经很吓人了，更别说制备病毒。人们不禁会问，这安全吗？其实科学家早已准备好各种防范措施，比如将病毒复制所需的基因和关键调控元件分离，这样它们就不会被包装成病毒颗粒，不受控制地感染和复制。同时，使用适当的生物安全设备以及处理病毒的标准做法，能减轻病毒载体造成伤害的机会。这些复制缺陷型的载体已逐渐成为标准工具，来执行细胞内成像、基因编辑和蛋白生产的任务。
研究人员可以从多家供应商购买到现成或定制的载体。Vector Biolabs的首席运营官X.D. Shao表示，它们通常是以病毒储液的形式提供的，可直接加入细胞或注射到动物中。由于腺病毒（与慢病毒和AAV不同）在特殊的包装细胞中可以复制，有时它也以病毒种子储液提供，这样科学家就能够不断产生载体。
另一种获得病毒载体的方法是购买质粒，它包含必需的调控元件和启动子（在某些情况下可诱导），以及可选的标签，协助用户观察和纯化所产生的病毒颗粒。许多质粒是立即可用的，包含特异的目的基因（GOI），或者为RNAi、shRNA或CRISPR/Cas9应用而设计。这些质粒可以是现成的产品，也可以从不同的公司订购，包括Vector Biolabs、GeneCopoeia和Cell Biolabs。
Shao认为，制备病毒载体需要对分子生物学、细胞生物学以及病毒学有一定的了解，如果实验室内部有这方面的知识，他们肯定能够自己制备病毒。多家公司的试剂盒都提供了所需的一切。
摘要：
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
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据斯克里普斯研究所（Scripps Research Institute）免疫学和微生物学教授John Elder介绍，病毒载体的存在已有数亿年。现代的实验室已经驯服了病毒，确保它既安全又有效，同时利用其能力来转导高比例的细胞。
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
制备病毒
利用病毒作为载体就已经很吓人了，更别说制备病毒。人们不禁会问，这安全吗？其实科学家早已准备好各种防范措施，比如将病毒复制所需的基因和关键调控元件分离，这样它们就不会被包装成病毒颗粒，不受控制地感染和复制。同时，使用适当的生物安全设备以及处理病毒的标准做法，能减轻病毒载体造成伤害的机会。这些复制缺陷型的载体已逐渐成为标准工具，来执行细胞内成像、基因编辑和蛋白生产的任务。
研究人员可以从多家供应商购买到现成或定制的载体。Vector Biolabs的首席运营官X.D. Shao表示，它们通常是以病毒储液的形式提供的，可直接加入细胞或注射到动物中。由于腺病毒（与慢病毒和AAV不同）在特殊的包装细胞中可以复制，有时它也以病毒种子储液提供，这样科学家就能够不断产生载体。
另一种获得病毒载体的方法是购买质粒，它包含必需的调控元件和启动子（在某些情况下可诱导），以及可选的标签，协助用户观察和纯化所产生的病毒颗粒。许多质粒是立即可用的，包含特异的目的基因（GOI），或者为RNAi、shRNA或CRISPR/Cas9应用而设计。这些质粒可以是现成的产品，也可以从不同的公司订购，包括Vector Biolabs、GeneCopoeia和Cell Biolabs。
Shao认为，制备病毒载体需要对分子生物学、细胞生物学以及病毒学有一定的了解，如果实验室内部有这方面的知识，他们肯定能够自己制备病毒。多家公司的试剂盒都提供了所需的一切。
把基因表达的工作交给病毒载体[选购宝典]
摘要：
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
分享到：
按部就班
典型的流程是：将你想要表达的GOI或想要干扰的RNA克隆到质粒载体上，进行某种筛选，如抗生素耐药性。随后用质粒转化细菌、铺板，并挑选出阳性克隆，确认包含GOI。接着就是扩增，并收集质粒。下一步是将这个质粒与其他一些质粒一起共转染细胞，通常是HEK细胞的衍生物。
慢病毒会分泌到上清中，“你只需要收集上清，并直接使用”，GE Healthcare的Dharmacon研发部门的资深科学家Melissa Kelley谈道。上清中通常含有细胞碎片，而滴度也不是很高，因此许多研究人员会进行纯化和浓缩。这取决于你的应用以及细胞的敏感性。对于AAV和腺病毒，病毒的收集需要对细胞进行反复冻融，接着是低速离心。
浓缩和纯化病毒的标准方法是超速离心，不过这耗时耗力，通量也低。Vectalys的CEO Pascale Bouillé认为，这不是一个工业过程。这家公司提供即用型的慢病毒颗粒，滴度在每毫升109个颗粒。对于那些打算用在体内的载体，他们还会增加一步层析纯化。Bouillé提醒说，并非所有的公司都提供相同纯度或滴度的载体，客户也要注意滴度测定的方式。
那些自己生产病毒载体的实验室也能使用一些公司的纯化试剂盒，包括Takara Clontech、Cell Biolabs、Applied Biological Materials和Sartorius。Bouillé认为这些最适合需要少量载体的科研用户。
如何选择
这三种载体类型有许多相似之处，包括它们能转导静止和分裂的细胞，以及能感染大多数的细胞类型。不过，它们之间也有几个主要的不同点。慢病毒整合到细胞的基因组，而腺病毒和AAV通常是游离的。对于腺病毒和AAV而言，前者主要应用在体外，而后者应用在体内。一般来说，AAV只能接受小的插入片段，大约在4.5 kb，但腺病毒能包装8 kb，而慢病毒则达到10 kb。
Kelley认为，慢病毒和腺病毒的选择在于实验的要求。例如，慢病毒能够产生稳定的细胞系，而不需要筛选；然而，它也存在整合的风险，可能导致有害突变。另一方面，腺病毒会表达其他基因。这是它在体内应用中不受欢迎的原因。
科学家们试图更好地了解基因表达的体内调控机制，而神经细胞、干细胞和原代细胞就成了必不可少的工具。然而，这些细胞难以用传统的方法转染。如果你想要检查GOI的表达，但无法让DNA进入你的细胞，那么别害怕。把它交给病毒吧！
（作者：Josh P. Roberts/生物通编译）
摘要：
多年来，这一领域主要围绕着三种病毒：腺病毒（Ad）、腺相关病毒（AAV）和慢病毒（lenti），每一种都有其自己的优势和不足。下面，我们就来了解一下病毒载体的各个方面，每种类型有哪些特征，以及它们最适合哪些应用。
分享到：
按部就班
典型的流程是：将你想要表达的GOI或想要干扰的RNA克隆到质粒载体上，进行某种筛选，如抗生素耐药性。随后用质粒转化细菌、铺板，并挑选出阳性克隆，确认包含GOI。接着就是扩增，并收集质粒。下一步是将这个质粒与其他一些质粒一起共转染细胞，通常是HEK细胞的衍生物。
慢病毒会分泌到上清中，“你只需要收集上清，并直接使用”，GE Healthcare的Dharmacon研发部门的资深科学家Melissa Kelley谈道。上清中通常含有细胞碎片，而滴度也不是很高，因此许多研究人员会进行纯化和浓缩。这取决于你的应用以及细胞的敏感性。对于AAV和腺病毒，病毒的收集需要对细胞进行反复冻融，接着是低速离心。
浓缩和纯化病毒的标准方法是超速离心，不过这耗时耗力，通量也低。Vectalys的CEO Pascale Bouillé认为，这不是一个工业过程。这家公司提供即用型的慢病毒颗粒，滴度在每毫升109个颗粒。对于那些打算用在体内的载体，他们还会增加一步层析纯化。Bouillé提醒说，并非所有的公司都提供相同纯度或滴度的载体，客户也要注意滴度测定的方式。
那些自己生产病毒载体的实验室也能使用一些公司的纯化试剂盒，包括Takara Clontech、Cell Biolabs、Applied Biological Materials和Sartorius。Bouillé认为这些最适合需要少量载体的科研用户。
如何选择
这三种载体类型有许多相似之处，包括它们能转导静止和分裂的细胞，以及能感染大多数的细胞类型。不过，它们之间也有几个主要的不同点。慢病毒整合到细胞的基因组，而腺病毒和AAV通常是游离的。对于腺病毒和AAV而言，前者主要应用在体外，而后者应用在体内。一般来说，AAV只能接受小的插入片段，大约在4.5 kb，但腺病毒能包装8 kb，而慢病毒则达到10 kb。
Kelley认为，慢病毒和腺病毒的选择在于实验的要求。例如，慢病毒能够产生稳定的细胞系，而不需要筛选；然而，它也存在整合的风险，可能导致有害突变。另一方面，腺病毒会表达其他基因。这是它在体内应用中不受欢迎的原因。
科学家们试图更好地了解基因表达的体内调控机制，而神经细胞、干细胞和原代细胞就成了必不可少的工具。然而，这些细胞难以用传统的方法转染。如果你想要检查GOI的表达，但无法让DNA进入你的细胞，那么别害怕。把它交给病毒吧！
（作者：Josh P. Roberts/生物通编译）
The CAR T-Cell Race
Tumor-targeting T-cell therapies are generating remarkable remissions in hard-to-beat cancers—and attracting millions of dollars of investment along the way.
| April 1, 2015
BUILDING A BETTER T-CELL: By modifying T cells to express chimeric antigen receptors (CARs) that recognize cancer-specific antigens, researchers can prime the cells to recognize and kill tumor cells that would otherwise escape immune detection. The process involves extracting a patient’s T cells, transfecting them with a gene for a CAR, then reinfusing the transfected cells into the patient.© LUCY READING-IKKANDALast December, scientists at Juno Therapeutics reported at the American Society of Hematology (ASH) meeting that, in an ongoing Phase 1 trial, its chimeric antigen receptor (CAR) T-cell therapy, JCAR015, put 24 of 27 adults with refractive acute lymphoblastic leukemia (ALL) into remission, with six patients remaining disease free for more than a year (ASH 2014, Abstract 382, 2014). This disease is extremely hard to treat and progresses rapidly when it becomes refractory; most patients die within a few months. “This response rate is unprecedented for patients who had stopped responding to all other treatments,” says Michel Sadelain, a founding director of Memorial Sloan Kettering’s Center for Cell Engineering and a cofounder of Juno.Founded just a year earlier, the Seattle-based company now has four CD19-targeting CAR T-cell therapies in trials. The premise is simple: extract a patient’s T cells from blood and train them to recognize and kill cancer by modifying them with a viral vector to express an artificial, or chimeric, receptor specific for a particular cancer-associated antigen—in this case, CD19, an antigen expressed in B-cell–related blood cancers—then reinfuse the cells back into the patient. (See illustration at left.) The engineered cells recognize and kill cancerous cells, while reactivating other immune players that have been dampened by cancer’s inhibitory signals. “CAR therapy is at the same time cell therapy, gene therapy, and immunotherapy,” says Sadelain. “It represents a radical departure from all forms of medicine in existence until now.” Promising preclinical results have moved Juno’s CD19 therapies into trials for ALL, non-Hodgkin’s lymphoma, and chronic lymphocytic leukemia (CLL), and the company has three more CAR T-cell immunotherapies for a number of solid cancers close behind.
A few weeks after the ASH meeting, Juno went public for a whopping $264.6 million, the largest biotech initial public offering (IPO) of 2014. Within a month, the company’s valuation rose from $2 billion to $4.7 billion, the largest among biotechs in a decade. By the end of 2016, the company plans to have 10 drug trials for six diseases up and running using CAR T cells produced in a brand-new manufacturing facility.
And Juno is not alone. This relatively new sector is experiencing a frenzy of scientific activity, corporate partnering, and financing that took off in late 2013, continued throughout 2014, and moved straight into the new year with no sign of letting up. By now, most major pharmaceutical companies have jumped into the CAR T-cell arena. In the past two years alone, at least half a dozen companies have made deals worth hundreds of millions of dollars up front, with much more expected in the future as products move through the pipeline. (See chart below.) This influx of funding is now supporting dozens of clinical trials.
While most of these studies are currently aimed at late-stage disease for which other therapeutic options have failed, researchers in the field anticipate that these immunotherapies could replace standard cancer treatments in the future. “While we are evaluating these therapies in advanced cancer now, we absolutely believe that they have the potential to become frontline therapies,” Sadelain says.
Long time coming
CAR T-cell therapy has had a lengthy run-up to what may appear to be overnight success. The first CAR T cells were developed at the Weizmann Institute of Science in Israel in the late 1980s by chemist and immunologist Zelig Eshhar. In 1990, Eshhar took a year-long sabbatical to join Steven Rosenberg at the National Institutes of Health, where they prepared CARs that targeted human melanoma. “We designed CAR T cells to overcome a number of problems in getting T cells to attack cancer,” says Eshhar. These problems included a tumor’s ability to escape immune recognition by silencing the major histocompatibility complex molecules and the generally immunosuppressive tumor microenvironment.
NEW AND IMPROVED CARs: Zelig Eshhar and Steven Rosenberg constructed the first CAR T cells using a modular design, including a specific cancer-targeting antibody outside the cell, a transmembrane component, and an intracellular costimulatory signaling domain that amplifies the activation of the CAR T cells. Second- and third-generation CAR T-cell technologies have added additional costimulatory domains within the cells, as well as additional receptors to improve targeting of the T-cell attack and minimize side effects.© LUCY READING-IKKANDAEshhar and Rosenberg constructed the CAR T cells with a modular design that included a specific cancer-targeting antibody, and later added a costimulatory signaling domain that amplifies the activation of the cells, giving them a stronger signal to multiply and kill cancer cells. Since that early work, researchers in both academia and industry have developed and tweaked each section of the modular design. (See illustration above.) “Ultimately, we needed 20 years to learn how to supercharge these cells to deliver anticancer activity,” says Arie Belldegrun, president and CEO of Kite Pharma in Santa Monica, California, which is assessing CAR T cells in six trials for B cell leukemia and lymphomas, and glioblastoma. Eshhar, a member of Kite Pharma’s scientific advisory board, continues to collaborate with Rosenberg, who serves as a special advisor to the company.
Juno is now working on two second-generation CAR technologies that incorporate mechanisms to further amplify T-cell activation or to dampen it, in the case of adverse reactions. (See “Safety concerns.”) These so-called “armored” chimeric antigen receptors are designed to combat the inhibitory tumor microenvironment by incorporating a signaling protein such as IL-12, which stimulates T-cell activation and recruitment. Juno believes “armored CAR” technology will be especially useful for solid tumors, whose microenvironment and potent immunosuppressive mechanisms can make raising antitumor responses more challenging.
Like Juno, Houston, Texas–based Bellicum Pharmaceuticals is working on refinements for next-generation CAR T-cell treatments. To better control antigen activation by its CAR T cells, for example, Bellicum is separating its dual costimulatory domain from the antigen-recognition domain, moving it onto a separate molecular switch that can be controlled by the small-molecule drug rimiducid. These T cells, known as GoCAR-Ts, can only be fully activated when they are exposed to both cancer cells and the drug.
In addition to altering the components of the CAR T cells themselves, researchers are also experimenting with different methods to introduce the receptors into the patients’ cells. At MD Anderson Cancer Center in Houston, Laurence Cooper and his colleagues are using a nonviral system called “Sleeping Beauty,” licensed from the University of Minnesota’s Perry Hackett, that relies on a transposon derived from fish to paste any desired gene into the genome. “This system employs electroporation [an electric current] to introduce elements of the Sleeping Beauty system into T cells,” says Cooper, who hopes the system will be less complex and cheaper to use than viral vectors.
While CAR T cells are being tested first as monotherapies, researchers are also giving thought to how best to use CAR T cells with other immunotherapies in the future. “We are excited about combining checkpoint inhibitors such as PD-1 [programmed death-1] inhibitors and anti-CTLA4 [anti-cytotoxic T-lymphocyte antigen 4] drugs with CAR T cells,” Eshhar says.
A frenzy of deal making
Over the past few years, the industry has been a hive of activity, with a half dozen companies forging deals valued at more than a half billion dollars in total. In addition to the perceived financial potential of these therapies, the feeding frenzy may in part be attributable to the fact that regulatory authorities are giving CAR T-cell treatments priority review for filling unmet medical needs. Many of these therapies are receiving orphan or breakthrough status from the US Food and Drug Administration (FDA), bringing expedited regulatory review, which translates into earlier realization of financial benefits from more rapid market entry. In November 2014, for example, the FDA granted orphan status to Juno’s JCAR015. Kite’s KTE-C19 for refractory aggressive non-Hodgkin’s lymphoma also recently received the designation from both the FDA and the European Medicines Agency. And the University of Pennsylvania /Novartis’s CTL019 for ALL received breakthrough status last July.CAR T-CELL DEALS
| Institution/Company | Date | Partner | Terms |
|---|---|---|---|
| University of Pennsylvania | August 2012 | Novartis | Undisclosed |
| Celgene | March 2013 | Bluebird Bio, Baylor College of Medicine | Unspecified upfront payment plus up to $225 million per product in option fees and milestone payments |
| Cellectis | June 2014 | Pfizer | $80 million upfront plus up to $185 million per product and royalties |
| Cellectis | January 2015 | Ohio State University | Undisclosed |
| Kite Pharma | January 2015 | Amgen | $60 million upfront and up to $525 million per product in milestone payments, plus royalties on sales and IP licensing |
| Md Anderson | January 2015 | Ziopharm, Intrexon | $100 million in stock and $15–20 million/year for 3 years |
Clinical results spark hope
In 2011, the Penn group described the results of an early trial of its CTL019 CAR T-cell treatment in three advanced chronic lymphocytic leukemia (CLL) patients (Sci Transl Med, 3:95ra73, 2011). The findings—including two patients who have now remained in remission 4.5 years after their treatment—served as an early demonstration that CAR T cells can successfully treat patients with late-stage disease. The team has now tested CAR T-cell therapies in about 125 people, with six different trials underway for pediatric and adult ALL, CLL, multiple myeloma, and non-Hodgkin’s lymphoma. Other CAR T-cell therapies are in trials for solid tumors, including ovarian, breast, and pancreatic cancers, and mesothelioma and glioblastoma.CAR T-CELL BIOTECH IPOs
| Company | Date | Value |
|---|---|---|
| Kite Pharma | June 2014 | $134.1 million |
| Bellicum | December 2014 | $160 million |
| Juno | December 2014 | $264.6 million |
| Cellectis | March 2015 | $228 million |
In a recent Penn study of 30 children and adults with relapsed or refractory ALL who received CTL019, 90 percent achieved total remission, and 78 percent were still living at the end of the study two years later (NEJM, 371:1507-17, 2014). Moreover, “very few patients—three—got a bone marrow transplant after CTL019, suggesting that this could be a replacement for bone marrow transplant and not just a bridge to transplant,” added senior author Stephan Grupp, director of translational research for the Center for Childhood Cancer Research at the Children’s Hospital of Philadelphia.
At ASH, Grupp discussed a follow-up study, including 39 pediatric patients, which showed a 92 percent complete remission rate following CTL019 treatment. Of those, 76 percent remain in complete remission after six months (ASH 2014, Abstract 380, 2014). “The first ALL patient treated is still in remission nearly three years later,” says Grupp. In September, Novartis pledged $20 million to build a Center for Advanced Cellular Therapeutics to manufacture CAR T cells on the Penn campus, to be completed next year. Penn is now conducting pilot trials aimed at solid tumors, including mesothelioma; ovarian, breast, and pancreatic cancers; and glioblastoma.
A team at the National Cancer Institute, including Rosenberg, has also reported successes with CAR T cell therapy, focusing on patients with refractory diffuse large B-cell lymphoma, an aggressive disease for which survival without treatment is measured in months. Following treatment with CD19-targeting T cells, 22 of 27 patients had either complete or partial remissions; 10 have remained cancer free for up to 37 months (ASH 2014, Abstract 550, 2014). These and other trials have demonstrated that CAR T-cell therapies can successfully treat leukemias and lymphomas in some patients for whom there are no other treatments.
There are currently many other CAR T-cell trials underway in leukemia and lymphoma, and more beginning in the near future. Scientists in this arena are energized by these and other results, and many see CAR T-cell therapies as the future of cancer treatment. “I believe these trials indicate that chemotherapy may be on its way out,” says MD Anderson’s Cooper.
Vicki Brower is a freelance science writer living in New York City.
| SAFETY CONCERNS Despite the growing number and length of remissions using CAR T-cell therapy to treat leukemias and lymphomas, key challenges remain—first and foremost, safety. There have been a half-dozen treatment-related deaths in the University of Pennsylvania and Juno trials in the past few years that involve a major side-effect of CAR T-cell therapy called cytokine-release syndrome (CRS). T-cell activation causes the release of inflammatory cytokines, producing symptoms including high fevers, aches, hypotension, and, more rarely, pulmonary edema and neurologic effects such as delirium. Researchers tie the severity of what they call a “cytokine storm” to tumor burden—a patient’s total mass of cancer tissue or quantity of malignant cells. One hypothesis for this is that higher tumor burden seems to incite a stronger immune reaction. Moreover, the deaths have all occurred in adults, some of whom had serious underlying medical issues, and others who had undiagnosed infections. Interestingly, children seem relatively resistant to severe CRS and, when they get it, are more easily managed, says Michel Sadelain of Memorial Sloan Kettering and Juno. “Adults do not tolerate the treatment as well as children, in whom the cells differ in speed of action and persistence,” Sadelain says. Treatment with an anti-IL6 antibody, or in severe cases, corticosteroids, can mitigate a cytokine storm’s severity, as can dosing with lower numbers of CAR T cells. “In our trial [on diffuse large B-cell lymphoma], we saw that toxicity was reduced in patients who received low-dose chemotherapy rather than high-dose [prior to CAR T-cell treatment], and lower numbers of engineered T cells [than given previously],” says James Kochenderfer of the National Cancer Institute (NCI). Bellicum is partnering with the University of Leiden in the Netherlands and the NCI, among others, to develop “suicide switches,” or safety on-and-off switches that are incorporated into CAR T-cell candidates to control T-cell activation and proliferation. And Juno’s second-generation “armored” CAR technologies include mechanisms to dampen T-cell activation. “It will be important to find new ways to overcome toxicity of CAR T cells,” says the Weizmann Institute’s Zelig Eshhar. |
The description of EP Vantage has also been updated from "a financial analysis company" to "the editorial team at life science market intelligence firm Evaluate Ltd."
The Scientist regrets the errors.
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