Nature Medicine_眼疾治疗的未来：研究者热论干细胞疗法的最优发展道路

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本期 Nature Medicine 和 Nature Review Drug Discovery，非常值得一阅。4 _) i4 O# m. p( Q3 V8 \3 n0 W
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本篇新闻报道全文如下：2 C: {0 I7 x: u" @. M  N5 N
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An eye to the future: Researchers debate best path for stem cell–derived therapies3 h; Q. k* m7 i- P
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Shraddha Chakradhar
1 b8 v% E% u; ?- ]Nature Medicine 22, 116–119 (2016) doi:10.1038/nm0216-116
4 Q* p6 q) z& dPublished online 04 February 2016
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In 2013, Masayo Takahashi and her team received approval to begin a clinical trial with just two people who had age-related macular degeneration (AMD), an eye disease that results in vision loss. The trial aimed to test the safety of a new therapy: the implantation of retinal cells that had been derived from induced pluripotent stem (iPS) cells. These iPS cells would, in turn, come from the recipients' own skin cells, making the therapy autologous in nature and eliminating the need for the addition of immunosuppressants to prevent the person's immune system from rejecting the transplanted cells. In 2014, one of Takahashi's patients, a 70-year-old Japanese woman with AMD, became the first person in the world to receive a therapy derived from iPS cells. She was not given immunosuppressants, and more than a year later, she has shown no adverse effects from the procedure. And although the procedure was not designed to test efficacy, the woman's visual acuity, which had been declining, has since stabilized.! T1 a" q% L4 a% m  ]
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In 2015, however, when Takahashi, a project leader at the RIKEN Center for Developmental Biology in Kobe, Japan, and her team prepared to perform the procedure on the second person, they hit a stumbling block. As with the first recipient, a guidance committee within the Japanese Ministry of Health, Labour and Welfare (MHLW), which had approved the trial, wanted the team to screen the batch of iPS cells derived from the second person for possible mutations. “The first patient's cells were beautiful,” Takahashi says. There were no mutations in her iPS cells or in the differentiated retinal cells.
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8 G2 }: ~4 K' w3 TWhen it came to the second patient, however, genetic sequencing of the iPS cells revealed mutations in three different genes, one of which is categorized as an oncogene in the Catalogue of Somatic Mutations in Cancer (COSMIC). Still, Takahashi was not concerned. There was no evidence to suggest that cancer had ever developed in the retinal pigment epithelium (RPE), a sheet of cells that serves as the final product transplanted into a recipient. “COSMIC is not a perfect list. And there isn't strong evidence to suggest this one gene is an oncogene,” Takahashi says. Furthermore, she adds that the guidance committee overseeing the trial had never before mentioned that the presence of mutations in the derived cells was grounds for concern. “We didn't promise mutation-free cells; we only promised safe cells,” she says.
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& k3 V2 T" v- p- {) ?- gAs the first of its kind in the world, the trial generated substantial global interest, and many of those scientists not associated with it were citing concerns about the mutations. “The final product is what matters,” Takahashi says. “But people only care about the genetic change in the iPS cells.” She adds that a patient's original skin cell samples could have oncogenic mutations in them already, “but people don't check for safety in those cells.”
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, F. I' r2 B7 v; R3 mIn Takahashi's case, the trial was planned with counsel from two separate regulatory bodies: the MHLW and the Ministry of Education, Culture, Sport, Science and Technology (MEXT), which oversees preclinical research in Japan. The latter was familiar with Takahashi's preclinical research into iPS cells and therefore understood that the presence of mutations without a strong link to cancer in and of itself was not a cause for alarm. But the individuals on the MHLW's committee, which gives clearance to clinical trials, were less familiar with the technique. “The discussion [surrounding this trial] was separated between the two ministries, and we fell in the gap,” Takahashi says. Plagued by what she calls the confusion that ensued as a result, Takahashi announced in October 2015 that the trial in the second person would be halted.4 g$ b) ?( Z2 D# O4 E: g

; h+ o8 r% A7 h3 S% r7 }In the decade since the discovery that mature cells could be coaxed back into stem cells, regenerative-medicine researchers have worked on developing autologous stem cell–derived therapies such as Takahashi's, in which cells from an individual are reverted to stem cells before being differentiated into mature cells that are then transplanted back into the donor. Despite these efforts, no iPS cell therapy has yet received regulatory approval anywhere in the world. Given the news of the trial suspension, those working within this space are left wondering whether autologous therapies still represent a viable approach. The leading alternative uses allogeneic cells—stem cells from a donor other than the person receiving treatment—that are then differentiated into mature cells.+ D+ k# H" r. g' R

; a2 n4 i0 p1 O1 ]Price check
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8 e0 G% q- r% G7 L# G: S; Y$ gWhereas the RIKEN trial was halted because of potentially concerning genetic mutations, the leading argument against the pursuit of autologous iPS cell therapies is the projected cost associated with the therapy. For every person who wants the autologous version of the AMD treatment, iPS cell lines need to be created—a process that takes roughly three months, says Hardy Kagimoto, CEO of Healios, a Japanese company co-founded by Takahashi. These cell lines are then differentiated into retinal cells, a process that adds another four months to the production timeline. Much of the cost is associated with the storage and maintenance of these cells, so the added step of creating unique lines for each person, along with the consequent additional maintenance time, can drive up the cost. For the 70-year-old recipient of the first transplant in 2014, for instance, Takahashi's group had to create 30 cell lines. The quality and safety of each line varies, Takahashi says, so they made multiple cell lines to enable them to pick the best one.
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2 ^+ k. ^0 w! Q& U% F& |! S+ rA bigger cost driver, however, is the extensive amount of testing that is required to demonstrate the procedure's safety. Because each therapy is personalized, safety would need to be proven for each cell line. In Japan, for instance, the guidance committee required high-quality whole-genome sequencing, epigenetic testing and tests in immunocompromised mice to check for tumorigenicity. Takahashi estimates that the safety testing alone cost close to $500,000. In addition, the associated functional costs for both storage and equipment maintenance are also high.
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Ultimately, a single autologous treatment, if it were performed today, would cost nearly $1 million, according to Kagimoto. “From a commercial perspective, it is almost impossible to make this work,” Kagimoto says. “When it's going to cost one person a million dollars for this treatment, you can't really sell it.”
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And yet proponents of autologous therapy think that the high price is merely a side effect of the newness of the procedure. “You calculate the cost of therapy when you reach phase 3 studies, not phase 1,” says Kapil Bharti, an investigator at the US National Eye Institute. He hopes to begin a phase 1 trial of autologous therapy for AMD. “Once you get to phase 3, then you take steps to reduce costs surrounding manufacturing, personnel or other factors.”; q& c$ f. w. s: B" f0 |

$ @4 @5 M/ \7 k' u4 C. A) R" Y( vThe mass production value offered by allogeneic cells makes them an attractive approach for many groups, including Healios and the folks behind the London Project to Cure Blindness, as well as its US counterpart, the California Project to Cure Blindness. Healios has licensed the technology used by Takahashi's team at RIKEN, but it plans to pursue an allogeneic approach in which iPS cell lines created from a healthy donor will be differentiated into retinal cells for transplantation.
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, M! q6 C) q# \; L+ C% c: a6 eLast summer, the London Project to Cure Blindness team, led by Peter Coffey, did its first transplant of retinal cells using an allogeneic approach: human embryonic stem cells that had been procured from a stem cell bank were differentiated into RPE cells. Two people received a 6 × 3 millimeter patch of the retinal cells, and they continue to do well, says Coffey. He adds that safety and efficacy results will probably be announced later this year.
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Dennis Clegg, a developmental biologist at the University of California, Santa Barbara, says that he and his colleagues recently received approval from the US Food and Drug Administration (FDA) to begin a phase 1 trial that will similarly implant a patch of embryonic stem cell–derived retinal cells into 20 people with AMD. Unlike Coffey's group, which targets the wet type of the disease in which blood vessels leak fluid into the retina, Clegg's group will target the dry type of AMD, which affects nearly 90% of those with AMD and for which there are currently no approved therapies. When it comes to cost, Clegg says that keeping the therapy affordable is a main goal of the California Institute for Regenerative Medicine, which is the trial's main funding body. “One benchmark to consider might be the cost for treatment of the wet form of AMD, whether Lucentis, Avastin or Eylea,” Clegg says. “We would love to come in with a price like Avastin,” which currently costs around $50 per injection for as many as 12 injections per year. Clegg's therapy would be a one-time procedure, but he adds that the goal is to keep the price of the product as low as possible.6 J7 N2 X, d/ X5 o5 h
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: {' A' W0 F+ D5 L" Z$ NJust as the presence of mutations sparked controversy in the case of Takahashi's autologous therapy, the risk of mutations is not absent in allogeneic therapy. However, because autologous therapy includes an additional step of taking a person's cells and first turning them into iPS cells, researchers hypothesize that the added manipulation may also increase the risk of mutations arising. By contrast, allogeneic therapy offers the advantage of being able to screen the starting line of stem cells, which are identical, for safety before they differentiate into retinal cells. Consequently, researchers would not have to do what Takahashi did by creating 30 different cell lines to test for safety.* W/ ~: L$ P8 p. T7 o

) E* U3 ^$ t1 _$ m: x* }3 q: z“We are all here are to make sure that patient safety is of importance,” Bharti says. “We made nine lines and didn't see a single driver mutation.” But he acknowledges that the changes in cell type during the production process does pose a risk of mutations. As a result, his team will genetically sequence all the cell lines produced for autologous transfer and check for more than 220 noted oncogenes.
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1 J/ f2 {* U4 C: j1 i6 |Others think that a bigger safety concern for both autologous and allogeneic therapy is tumorigenicity, which could stem from the accidental transplantation of a batch of stem cells that has not fully differentiated into the final product. To gain FDA approval for a trial, in addition to demonstrating a lack of toxicity, researchers must show no tumorigenicity in the differentiated cell sample. “We were not required to do genomic sequencing of the embryonic stem cell line we were using,” Clegg says. It took the California team nine months to show a lack of tumorigenicity in their product. “We were required to put the human dose in a rat and let the animal live its whole life. “Then we had to slice and dice the tissues in the animal to make sure that there was no tumor. Can you imagine doing that for every autologous line you make?” Because there are currently no FDA-approved autologous trials for iPS-derived therapies under way—Bharti's trial in 2017, if approved, would be the first—it remains unclear whether the safety requirements for these trials will be different from those for allogeneic therapies such as Clegg's. The FDA, however, told Nature Medicine that it “neither requires, nor anticipates requiring, comprehensive genetic sequencing to be performed for iPS cells that are made from patient cells for autologous therapy.” Sarah Peddicord, a spokeswoman for the agency, qualified that there could be unique case-by-case circumstances in which the performance of genetic sequencing is recommended or deemed necessary.
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& Q7 ]0 V( ]8 M9 V2 e& [But there is no standard, even among those who are developing allogeneic therapies, when it comes to testing for mutations. Coffey's group in London did perform genetic tests on the embryonic stem cells before inducing them to differentiate, and the team plans to continue this testing when it expands its work to autologous therapies.6 Y( E% u0 b  a) E/ R1 R
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The question across the board, however, is what the presence of mutations in stem cells means. Takahashi is confident that, at least for retinal cells, there is no evidence that these mutations pose a risk.& L; a0 E  c/ I" {

- k# n5 ?$ g" }  gOthers also think that it is the safety profile of the final product that matters. “It's not a question of mutation, per se,” Coffey says. “Cells, when they are taken out [of the body] and put in a dish, will change their profile, but does that make them any less safe? We're not wishing to look at just potential, but whether it's really affecting the safety of the cell.”
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( k; a' `9 `' \+ `At least for now, no one seems to have an answer to this question. Some think that because these therapies are still in their early stages, proceeding with caution is safe, even if it means abandoning a batch of cells. Others, such as Kagimoto, assert that although the presence of mutations is of concern, it should not deter researchers from proceeding with the treatment. “Organ transplantations are considered daily, but [regulatory bodies] don't check for genetic mutations in organ donors, even though there is a risk there,” he says. Stem cell–derived transplants, he argues, should not be viewed any differently. He adds that Healios, however, will be testing for mutations in the cell lines that it generates.
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Researchers in the ocular regenerative space take solace in the fact that the eye is perhaps the best medium in which to catch cancers early. “The advantage of the eye is that we can look directly into the back of the eye and look for abnormal growth of cells very easily,” says Stephen Huhn, chief medical officer of StemCells, Inc., a biotechnology company that was developing an allogeneic therapy for ocular disorders. “If there were an abnormal reaction or growth of the cells, you'll see it simply by looking at the retina.”5 Z2 l, [& {6 H3 Z+ O# V8 l

; r; B! ]4 `7 n( [* DBalancing act
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3 J* {9 a  m9 l0 K# rWhereas the current cost and high mutation risk of autologous therapy seem to be deterrents, the lack of immune rejection helps to recommend the approach. Given that at their core, these procedures are transplants, the rejection of the cells by the host immune system is a concern. With autologous therapy, however, the implanted cells originate from the recipient's own body and so host rejection has not yet—albeit for the one 70-year-old person in Japan in 2014—been proven problematic. But this is not the case with allogeneic cells, because they do not originate from the person's body." N1 l7 v. B. Z' r0 R7 @

- _9 \5 n5 \, Z6 X  BWhen receiving allogeneic therapy, individuals are also given immunosuppressants, often in the form of steroids, to keep the immune system from attacking the newly implanted cells. The downside, according to some, is that immunosuppressants may be a long-term requirement. A continually compromised immune system could lead to the development of other diseases. However, this may not be case for the eye.0 N7 q( @# H# l/ p; A1 V; Q. x8 `

" w3 G8 W- U6 O" a  Y8 k“The eye is immune-privileged,” says Emile Nuwaysir, president of Cellular Dynamics, Inc. (CDI), one of the first companies to perform large-scale manufacturing of iPS cells for use in research. “Your heart, for example, is bathed in blood and the immune system it carries, but the eye is sealed and largely protected.” This means that the need for immunosuppressants is much more reduced for transplants involving the eye than those in other parts of the body. Individuals who are receiving allogeneic therapy need only short-term immunosuppressants, and some scientists, such as Lorenz Studer, think that when it comes to the eye, immunosuppressants can be done away with altogether. “Retinal cells have low immunogenicity, so short-term immunosuppressing is okay, but you'll be fine even without them,” says Studer, who is a researcher at New York's Memorial Sloan Kettering Cancer Center.9 v* d- T; \& P$ r+ A
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Studer is applying to the FDA for clearance for an allogeneic trial that will transplant young neurons into the brains of those with Parkinson's disease. The brain, similarly to the eye, is an immuno-privileged region of the body, and the hope is that innervating the striatum, the region of the brain into which the neurons will be transplanted, will help to restore some lost movement in these individuals. “We will transiently immunosuppress,” he predicts, “but we are not far away from not having to immunosuppress.”( c5 a, D" n; U/ J* a

$ k% O0 z) U" P- tCoffey's two patients received anti-inflammatory agents, and Clegg has stated that the people in his trial will also receive immunosuppressants. A trial conducted by Ocata Therapeutics of Marlborough, Massachusetts, tested the safety and tolerance of embryonic stem cell–derived retinal cells in four individuals, two of whom have AMD and two who have a related disease known as Stargardt's macular dystrophy. The team showed that despite having stopped immunosuppression four weeks after surgery, three of the people treated required additional immunosuppression, which possibly led to pneumonia in one individual (Stem Cell Reports 4, 860–872, 2015). Reduced need or not, no one thus far has stepped away from the administration of immunosuppressants.! I; j! {: m1 P; l5 Q4 ^

2 u6 p: H1 p/ _5 D: c  r& sAn approach that may offer the benefit of reduced rejection by the host immune system, as with autologous therapy, but may also allow for the large-scale safety testing that is done for allogeneic therapies, is also emerging: matching a person's human leukocyte antigen (HLA) subtype—protein markers that indicate immunocompatibility—with the HLA subtype of stem cells in a bank. This enables researchers to design a cell line that may be better tolerated by the recipient. Such an approach is already carried out for bone marrow and many organ transplants, where the rate of rejection by the host body is higher than in the eye, and now companies are hoping to extend this service for other parts of the body. CDI, for example, has begun to develop a bank dedicated to iPS cells that are classified by HLA subtype.
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“I personally think that the HLA paradigm combines the economics of allogeneic therapy with the matching potential of immunogenic approach,” Nuwaysir says.
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Similarly to how blood banks serve those who need transfusions, CDI hopes that its bank will serve as a resource for those who want to match individuals with stem cells needed for stem cell–derived therapies. The company has completed its pilot phase of recruiting and generating cell lines from five donors, and it hopes to populate the rest of the bank with nearly 200 more donors who will represent, in terms of HLA subtype, roughly 95% of the US population. CDI has partnered with Bharti's group and provided the materials for his anticipated 2017 trial.! ]8 N5 l% D/ X- Q; K* C, m

# y1 N! l# E8 G, ?Chris Parker, CDI's business officer, added that while people wait for the cost of autologous therapy to come down, the HLA model could serve as an efficient intermediate. Healios will largely be pursuing an allogeneic approach and hopes to bring a product to market by 2020. The company also plans to explore the HLA-matching approach as part of its development pipeline, according to Kagimoto. Takahashi, too, has been collaborating with Shinya Yamanaka, director of the Center for iPS Cell Research and Application (CiRA) in Kyoto, Japan, to develop therapies that utilize HLA matching, instead of relying on extracting cells from people for conversion to iPS cells. “We have been evaluating the immune reaction to HLA-matched retinal cells in monkeys,” Takahashi says. “These cells suppress the immune reaction naturally, and from the data so far, it seems we won't need any immunosuppression,” but they will continue to evaluate this approach for the next three years, she adds.
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. G0 `. c$ q1 fDespite the tremendous potential of autologous therapy, the limited success seen by those who are hoping to develop such a therapy casts doubt on whether it will prove viable. Takahashi told Nature Medicine that once reasonable social consensus is reached that the presence of mutations in itself is not a deterrent to therapy, she would like to restart the work with autologous transplantation. She also hopes that the launch of Japan's new Agency for Medical Research and Development, which aims to bridge the gap between the MEXT and the MHLW, will help to avoid future pitfalls.7 Z: D& s$ H' U
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Some, such as Studer, stand firm in their conviction that for the foreseeable future at least, allogeneic therapies are the better investment. “We can make billions of cells, lots of doses, test many times and ensure standardization,” he says. For others, the bigger concern is cost. “If cost is not a factor, then autologous is the way to go,” says Stephen Tsang, a researcher who is working on juvenile forms of macular degeneration at Columbia University. “If cost is a factor, then nonautologous is the way to go.”
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Still, few scientists currently pursuing allogeneic therapy are dismissive of the autologous approach. In fact, Coffey's group received a grant from the UK's Medical Research Council in early 2015 to run an autologous trial, which he hopes will begin sometime in 2017. “The beauty of the project here in London is that we will have both the [allogeneic] clinical trial data, and then soon after that, we will have the autologous trial data,” Coffey says. Having both sets of data will enable them to compare these two types of approaches, which will thus shed some light on the debate over which path to pursue. “It is the dream of the iPS field that it will be individualized medicine,” says Clegg, who served as a scientific advisor for Bharti's work. As far as which of the two main approaches to follow, he adds that “it's too early to tell at this point, and it's probably worthwhile to work towards both strategies.”
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7 u6 o' S3 M9 v# N& j“The problem is that there aren't enough answers,” Coffey says. “There's a lot of faith and hope, but no concrete answers yet.”