Stem Cells in Ophthalmology Update 5 Gene Defects Common in Induced Stem Cells

As the senior editor, John Gever, of MedPage Today reported, following the publication of three studies about induced pluripotent stem cells in the March 3rd, issue of Nature, “The road to regenerative medicine based on induced pluripotent stem cells (iPSCs) may have developed a giant pothole, with new studies showing that the cells are prone to several types of genetic defects.”

The three studies showed that the reprogramming process and subsequent culture of pluripotent stem cells in vitro can induce genetic and epigenetic abnormalities in these cells. The authors of the studies and the editorialist said that the results raise concerns over the implications of such aberrations for future applications of pluripotent stem cells.

Point mutations, copy number variations, and abnormal DNA methylation patterns all appear to crop up during generation of iPSCs. The frequency of such defects significantly exceeds what is normally found in human embryonic stem cells or in fibroblasts, the somatic cells from which iPSCs are usually derived.

"The studies raise concerns over the implications of such aberrations for future applications of iPSCs," wrote Martin F. Pera, PhD, of the University of Southern California in Los Angeles, in an accompanying editorial commentary. But he noted that it remains unknown whether the genetic "reprogramming" undertaken to generate iPSCs from fibroblasts is itself responsible for the genetic defects. Perhaps, Pera indicated, such defects "would be common to any experimental situation in which a cultured cell is subjected to strong selection and replication pressures in vitro."

Each of the three studies focused on a different type of genomic defect.

Kun Zhang, PhD, of the University of California San Diego, and colleagues from several other institutions looked at rates of point mutations in 22 iPSC lines and in the fibroblasts from which they were generated. The average number of mutations in protein-encoding genomic regions in each cell line was close to six. "Every single stem cell line we looked at had mutations. Based on our best knowledge, we expected to see 10 times fewer mutations than we actually observed," Zhang said.

All of the 22 iPSC lines had at least one "nonsilent" mutation that affected the resulting protein. One line showed 12 such alterations leading to mutated proteins. The mutations took different forms, the researchers reported, including splice variants and nonsense mutations.

In addition, they wrote, the abnormalities "were enriched in genes mutated or having causative effects in cancers."

Another of the Nature papers, by investigators from the Samuel Lunenfeld Research Institute in Toronto and elsewhere, examined gene copy-number variations that arise in iPSCs. Samer Hussein, PhD, of the Lunenfeld Institute, and colleagues found that these variations -- which included deletions as well as multiple copies -- were present in 37% of the iPSC lines they analyzed. Such variations were present in just 15% of fibroblasts and 25% of human embryonic stem cells (ESCs). The defects may be less significant in practical applications, however, relative to the point mutations. Hussein and colleagues reported that the highest rates of copy-number variations in iPSCs were seen in early-passage cells, whereas rates dropped with additional passages.

"Most of these novel CNVs rendered the affected cells at a selective disadvantage," the researchers explained. "Expansion of human iPSCs in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ESCs."

The third paper investigated what authors Joseph Ecker, PhD, of the Salk Institute for Biological Studies in La Jolla, Calif., and colleagues called "aberrant epigenomic reprogramming."

DNA methylation patterns help regulate gene expression and are variable during life as well as transmissible during reproduction. Ideally, causing an adult cell to revert to a stem-cell state would also involve reestablishing stem cell-like methylation patterns. Ecker and colleagues therefore generated whole-genome methylation profiles of five iPSC lines along with undifferentiated human ESCs, somatic cells, and differentiated iPSCs and ESCs.

They found that the reprogramming of methylation patterns in iPSCs was generally successful when looked at across the entire genome. "Overall, this process generates an iPSC methylome that, in general, is very similar to that of ESCs," they wrote. But in their base-by-base analysis, Ecker and colleagues discovered hundreds of differentially methylated regions compared with ESCs. Some of these were on the megabase scale, which apparently were "repeatedly resistant to reprogramming."

There were detectable differences in cell appearance or function as a result, the researchers noted. Moreover, whereas cells with copy-number variations failed to survive multiple passages, the abnormal methylation patterns did not appear to affect cell survival, as differentiated iPSCs retained the abnormal patterns.

That these aberrations "cannot be erased by passaging and are frequently transmitted through cellular differentiation has immediate consequences for the derivation and use of iPSCs," Ecker and colleagues warned.

In his "News and Views" commentary, Pera pointed out that these are not the first studies to warn of genomic irregularities in iPSCs. Two previous analyses, one published in 2010 and another earlier this year, also documented abnormal chromosome numbers and gene copy-number variations in the cells. These and the new Nature studies raise a number of questions about the future of iPSC research, Pera contended. Perhaps the most important, he wrote, "is the biological significance of the changes."

He indicated that missing or duplicated chromosomes would clearly disqualify cells from use in therapy, as would a high frequency of mutations in genes associated with cancer or known genetic disorders. "However, the many subchromosomal changes, copy-number variations, or point mutations that are not obviously associated with known disease-related genetic abnormalities pose challenges to interpretation," Pera argued.

He suggested that high-throughput functional genomics may be the best approach to resolving the problems -- which need to be addressed before iPSCs can become a basis for human disease treatments.

All three studies were supported by government and foundation grants. No commercial funding was reported.

The three studies are:

Gore A, et al, "Somatic coding mutations in human induced pluripotent stem cells" Nature 2011; 471: 63-67.

Hussein S, et al, "Copy number variation and selection during reprogramming to pluripotency"  Nature 2011; 471: 58-62.

Lister R, et al, "Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells" Nature 2011; 471: 68-73.

And the accompanying editorial:

Pera M, "The dark side of induced pluripotency" Nature 2011; 471: 46-47

Source:
MedPage Today, John Gever, Senior Editor
March 2, 2011

In addition, Healthzone Canada published a lengthy review of the Toronto study: “Toronto Scientists Report Roadblock in Stem Cell Field”; and a press release from the UC San Diego Health System“Mutations Found In Human Induced Pluripotent Stem Cells”, reported in depth on the study from the scientists and colleagues from the University of California, San Diego:

For more on “induced pluripotent stem cells “(iPSCs) and the three other types – "embryonic stem cells" (embryonic SCs, or human embryonic stem cells hESCs), "adult stem cells" (adult SCs) and "parthenogenetic stem cells" (hpSCs), please see my Primer on the Use of Stem Cells in Ophthalmology.

Stem Cells in Ophthalmology Update 12 Updated Table of Company Participants

When I first wrote about stem cells in ophthalmology (Primer) in September 2010, I was able to identify six companies participating. Since then, and with the help of a few friends, I can now identify eleven companies using stem cells to treat ophthalmic diseases. Since I believe access to knowledge is very important, here is my revised table of companies using stem cells in treating ophthalmic diseases.

Please let me know of any corrections or omissions.

(An easier to read pdf file of this table is available from the author via email request.)

Stem Cells in Ophthalmology Update 26 First Wet AMD Patient Treated With RPE Derived from iPS Cells

Earlier this week, it was reported that Masayo Takahashi, an ophthalmologist at the RIKEN Center for Developmental Biology (CDB) in Kobe had appeared in front of a 19-member health-ministry committee for the safety of the clinical use of stem cells. She was flanked by Shinya Yamanaka, the biologist who first created iPS cells. Yamanaka shared the 2012 Nobel Prize in Physiology or Medicine for his breakthrough and now heads the Center for iPS Cell Research and Application in Kyoto. Takahashi was seeking approval to implant a retinal pigmented epithelial (RPE) sheet made from induced pluripotent stem (iPS) cells into a human patient.

Takahashi and her collaborators had shown in monkey and mice studies that iPS cells generated from the recipients' own cells did not provoke an immune reaction that causes them to be rejected. There had been concerns that iPS cells could cause tumours, but Takahashi's team found that to be unlikely in mice and monkeys.

To counter further fears that the process of producing iPS cells could cause dangerous mutations, Takahashi's team had performed additional tests of genetic stability. Guidelines covering the clinical use of stem cells require researchers to report safety testing on the cells before conducting transplants. The health ministry said that no problems were found and that the human trial could commence.

Only four days later (Friday, September 12th), the first patient was treated with the implanted sheet of RPE cells. She derived them from the patient's skin cells, after producing induced pluripotent stem (iPS) cells and then getting them to differentiate into retinal cells.

This is a major first for the stem cell and regenerative medicine fields.

Takahashi and her collaborators have been using induced pluripotent stem (iPS) cells to prepare a treatment for age-related macular degeneration. Unlike RPE derived from embryonic stem cells (i.e., as being done by Advanced Cell Technology), iPS cells are produced from adult cells, so they can be genetically tailored to each recipient. They are capable of becoming any cell type in the body, and have the potential to treat a wide range of diseases. The CDB trial will be the first opportunity for the technology to prove its clinical value.

A Japanese woman in her 70s is the world's first recipient of cells derived from induced pluripotent stem cells, a technology that has created great expectations since it could offer the same advantages as embryo-derived cells but without some of the controversial aspects and safety concerns.

In a two-hour procedure starting at 14:20 local time, a team of three eye specialists lead by Yasuo Kurimoto of the Kobe City Medical Center General Hospital, transplanted a 1.3 by 3.0 millimeter sheet of retinal pigment epithelium cells into an eye of the Hyogo prefecture resident, who suffers from age-related macular degeneration.

The procedure took place at the Institute of Biomedical Research and Innovation Hospital, next to the RIKEN Center for Developmental Biology (CDB) where ophthalmologist Masayo Takahashi had developed and tested the epithelium sheets.

Afterwards, the patient experienced no effusive bleeding or other serious problems, as reported by RIKEN.

Another important element to this story is that Japan has a clinical translation pipeline that is now faster with recent changes in regulations than that of the US. For example, this and future iPS cell-based transplants were approved as part of a clinical study, a type of clinical research mechanism that doesn't exist in the US. It is safe to say that the same technology with the same research team and outstanding level of funding would still be at least a few years away from their first patient in the US due to the different regulatory scheme.

As noted by Dr. Paul Knoepfler, in his writeup about the procedure:

 “The patient is clearly a brave hero. The team transplanted a huge (from a bioengineering perspective) 1.3 x 3.0 mm sheet of RPEs into the retina of the patient, who did not have any clear immediate side effects from the procedure. Keep in mind again that this sheet was made indirectly from the patients own skin cells so it is an autologous (or self) transplant, a notion that 10 years ago would have seemed entirely like sci-fi.”

“This is not only a huge milestone, but also an astonishingly fast translation of iPS cell technology from the bench to the bedside.”

 “Also, on the positive side we have the encouraging results from the ongoing clinical trials from Advanced Cell Technology (ACT) using a similar approach to macular degeneration, but employing human embryonic stem cells to make the RPEs.”

“For the vision impaired and the broader stem cell field, it is heartening to have two such capable teams working to cure blindness with pluripotent stem cells.”

Sources:

1.Next-generation stem cells cleared for human trial: Japanese team will use 'iPS' cells to treat patient with degenerative eye disease, Nature, David Cyranoski, September 10, 2014.

2. Japanese woman is first recipient of next-generation stem cells: Surgeons implanted retinal tissue created after reverting the patient's own cells to 'pluripotent' state, Nature, David Cyranoski, September 12, 2014.

3. Stem cell landmark: patient receives first ever iPS cell-based transplant, Knoepfler Lab Stem Cell Blog, Paul Knoepfler, Septermber 12, 2014.

Gene Therapy in Ophthalmology Update 21 New Gene Therapy Company Spark Therapeutics Launches

Children’s Hospital of Philadelphia (CHOP) announced that it had spun off its work in gene therapy to a new, fully integrated company, Spark Therapeutics, that will assume control over two current gene therapy clinical trials: a Phase III study for Leber’s Congenital Amaurosis, an inherited disease that results in blindness caused by mutations of the RPE65 gene, and a Phase I/II study for hemophilia B. The new company is also advancing toward the clinic with gene therapy programs to address neurodegenerative diseases and additional hematologic disorders and other forms of inherited blindness. One such program, in the latter category, already in pre-clinical development at CHOP, could be its study for the treatment of Choroideremia, a rare inherited disorder that causes progressive loss of vision due to degeneration of the choroid and retina.

Editors Note: It should be noted that one clinical trial using gene therapy to treat Choroideremia is already underway at Imperial College London and Oxford University, in conjunction with Moorfields Hospital in London.

The new company has been launched with a $50 million capital commitment from Children’s Hospital to advance and commercialize multiple ongoing programs with clinical proof of concept.

As noted by Susan Young, writing about the launch in Technology Review, “Spark has a chance to be the first gene-therapy company to obtain FDA approval. Results for a late-stage trial of a gene therapy for Leber's Congenital Amaurosis ... are expected by mid-2015. That treatment is one of several gene therapies in or nearing late-stage testing contending to be the first gene therapy approved by the FDA for sale in the U.S.”

The Phase III trial was initiated late last year, and CHOP has made significant progress in enrolling patients. Spark will be sharing additional details on its progress and encouraging results in the very near future.

And, as shown in my table of Ongoing Clinical Trials in Ophthalmology, it is the only gene therapy trial (in ophthalmology) that has advanced to Phase III.

Editors Note: For clarity, is should be noted that there are five other clinical trials underway to treat Leber’s, as shown in my table, but all are currently Phase I or Phase I/II studies. The Spark Therapeutics trial is the farthest advanced.

“The creation of Spark is the culmination of a decade-long commitment by CHOP and our founding team to drive the field of gene therapy forward during a time when many in the industry had moved away,” said Jeffrey D. Marrazzo, co-founder, president and chief executive officer of Spark Therapeutics. “Their vision and long-term dedication have enabled us to effectively address many of the key challenges facing the field and to emerge with one of the industry’s most robust clinical-stage gene therapy pipelines; as well as exclusive rights to commercialize a proprietary manufacturing platform, supply from a world-class manufacturing facility and a founding team with a proven track record of executing safe and effective gene therapy trials for nearly two decades. We are working with great urgency and care to deliver gene therapy products with the potential to transform the lives of those affected by severe genetic diseases.”

Spark builds on the work of CHOP’s Center for Cellular and Molecular Therapeutics (CCMT), established in 2004 as a world-class center for gene therapy translational research and manufacturing. Many of the CCMT’s leaders will assume management roles within Spark or engage with the company as scientific advisors, including Katherine A. High, M.D, a gene therapy pioneer who has served as the director of the CCMT since its inception.

“Gene-based medicines are among the most complex therapeutics ever developed,” said Dr. High. “We at CCMT have persevered through more than a decade of scientific and clinical development and are now closer than ever to realizing the ambitious vision of one-time, potentially curative therapies to address serious genetic conditions. The team at Spark has incredible goals for the treatment of diseases including hemophilia B and inherited blindness, and we look forward to working with them to deliver groundbreaking new treatments to patients in need.”

Spark has entered into agreements with multiple academic institutions to assemble the technology, programs and capabilities needed to deliver its pioneering gene therapy products. Notably, Spark has exclusive rights to commercialize CHOP’s proprietary manufacturing technology and will use clinical-grade gene therapy vectors produced by the CCMT’s state of the art good manufacturing practices (cGMP) clinical facility.

Pioneers in AAV delivery

Over the past two decades, the Spark leadership team has developed unrivaled expertise in the design, manufacturing and delivery of gene therapies using adeno-associated virus (AAV) vectors. AAV has been demonstrated in clinical studies to be a safe and effective vehicle for the delivery of genetic material into targeted cells and provides unique advantages over alternative delivery approaches. The Spark team was among the first to demonstrate human clinical proof of concept in two distinct organ systems — the eye and the liver — establishing a strong foundation for the company’s current programs, and has clinical experience in 15 studies across diverse genetic and non-genetic diseases and five distinct routes of administration.

Spark’s most advanced clinical program is a Phase III study to address blindness caused by mutations in the RPE65 gene. There is currently no pharmacologic treatment for this form of inherited retinal degeneration, which ultimately causes irreversible blindness.

The open-label, randomized, controlled study builds on an earlier clinical study in which 12 patients with RPE65-related blindness demonstrated notable improvement in visual function, moving in some cases from being profoundly blind to being able to recognize faces and ambulate independently. All school-age patients enrolled in the trial were able to transfer from Braille classrooms to sighted classrooms.

One such patient was Corey Haas, whose story is related in the book “The Forever Fix: Gene Therapy and the Boy Who Saved It”.

Corey Haas, his parents, and the CHOP team that treated his Leber’s and gave him back his vision.

 

Read Corey’s story in Ricki Lewis’ book, The Forever Fix: Gene Therapy and the Boy Who Saved It.

The team's experience in the clinical study of gene therapy – from designing and manufacturing vectors to conducting studies that have shown strong potential for safety and efficacy – is unparalleled in the field. Clinical-grade vectors prepared by the team have been used to safely treat more than 100 human subjects in 12 clinical trials in the U.S. and EU, across five parenteral routes of administration in genetic and non-genetic diseases. No other group can claim this breadth of expertise and experience in human gene therapy.

The adeno-associated virus (AAV) vectors used in the clinical programs have been demonstrated to be safe and effective vehicles for delivering genetic material into targeted cells, providing unique advantages over alternative therapeutic approaches. The team has established human proof of concept in two organ systems – the eye and the liver – and are advancing a Phase III program in blindness caused by mutations of the RPE65 gene; a Phase I/II program in hemophilia B; and preclinical programs in neurodegenerative diseases and other hematologic disorders and forms of inherited blindness.