Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Cellular therapy—the introduction of live cells for the purpose of improving a patient’s health—is hot. Just witness bone marrow transplantation and the various immunotherapy treatments for cancer. Most cell therapy today is conducted using adult cells, but as stem cell derivation and differentiation technologies mature, much attention is also being focused on the potential of pluripotent stem cells (PSCs) for cell therapy.
There are currently at least two active clinical trials involving PSC-derived cells, with several others moving toward regulatory approval, says Jeanne Loring, professor and director for the Center for Regenerative Medicine at The Scripps Research Institute.
Why iPSCs?
Induced pluripotent stem cells (iPSCs) are largely seen to be the functional equivalent to embryonic stem cells (ESCs), but they are easier to obtain and are devoid of much of the ethical and regulatory baggage associated with derivation of ESCs.
iPSCs can, theoretically, be derived from any individual, male or female, of any age and state of health (even cadavers)—imbuing the cells with the genetics of the donor.
In theory, iPSCs can be grown indefinitely, and thus to any quantity, and differentiated into any cell type in the body.
“The reason this is exciting to everyone is because we never had human cells of these specific cell types before—we only had cell lines or animal cells as the equivalent,” remarks Mahendra Rao, vice president for research for the New York Stem Cell Foundation.
Adult stem cells, such as hematopoietic stem cells or mesenchymal stem cells, for example—when economically viable and available in sufficient quantities—would be Rao’s first choice for cell therapy. “But for a large number of cell types and indications, we don’t have an adult source.”
Making iPSCs
Shinya Yamanaka won the Nobel Prize for his 2006 discovery that by using a few specific transgene products—now often termed Yamanaka factors—cells could be reprogrammed back to an essentially embryonic state.
Yamanaka originally used retroviruses to make iPSCs, which express for a short time and then generally become silenced. “The problem is that retroviruses integrate randomly, and so sometimes they land at a place where they just don’t silence; they may also integrate in a place that causes a mutation, such as into a tumor suppressor gene, and you don’t want cells that are prone to generate tumors,” explains Michael Kyba, associate professor of pediatrics at the University of Minnesota. “So you’d really rather not mess at all with having anything integrated into the iPSC.”
The second generation of reprogramming efforts used episomal plasmid vectors, and the current (third) generation is RNA-based, says Nick Asbrock, product manager for pluripotent and differentiated cell technologies at MilliporeSigma.
Episomal vectors “don’t mean to integrate, but they do integrate,” points out Loring. “The length of the episome that actually integrates is different. People have to check.”
RNA can be delivered directly to the cells in a variety of ways. For example, individual Yamanaka factor mRNAs, or pools of these, can be purchased as kits from a variety of vendors.
Several companies offer kits that feature single-transfection polycistronic RNA coding for all the Yamanaka factors. “It’s engineered to self-replicate, so over about a 15-day period, the RNA will over-express the proteins that are engineered into the Simplicon strand,” says Asbrock, referring to MilliporeSigma’s Simplicon™ RNA Reprogramming Kit, the first such product to market when introduced two years ago. With non-self-replicating RNA, “you actually had to transfect every day for upwards of seven to 10 days … because they would degrade within a day,” shares Asbrock.
The option used by Kybaand Loring of late is the CytoTune™-iPS 2.0 Sendai Reprogramming Kit from Thermo Fisher Scientific. “The virtue of Sendai virus is that it’s an RNA virus that replicates through an RNA intermediate—there’s no DNA involved,” says Kyba, explaining that “whenever you put DNA into cells, you worry that some of the DNA is going to end up integrated in the genome.” (“And it’s manufactured without anything the FDA considers dangerous,” adds Loring.) The downside? “It’s quite expensive,” Kyba laments.
Booster kits are available for more recalcitrant cells. MilliporeSigma’s kits use proprietary small molecules, for example, while Stemgent’s uses microRNA, to facilitate and speed reprogramming.
For researchers who prefer to begin above the ground floor, iPSCs from male and female, diseased, healthy and at-risk donors can be purchased off the shelf through the Coriell Institute for Medical Research.
iPSCs on trial
Loring’s proposed clinical trial will reprogram Parkinson’s Disease patients’ own (autologous) cells, differentiate these into dopamine-producing (TH+) neurons and introduce them back into the patient. “We know that at some point those people won’t have any dopamine cells remaining in the right part of their brains.” Autologous cells alleviate most worries about immune rejection; introducing allogeneic cells (those from a different donor), even into a privileged site such as the brain or the eye, necessitates the patient undergo immunosuppression.
Loring’s group puts great emphasis on quality control (QC). At each stage of the reprogramming and differentiation process, it performs whole genome sequencing, looks at transcription with RNA-Seq or arrays and conducts “DNA methylation analysis to get an idea of what’s going on in the epigenome.”
Because only 30,000 introduced TH+ cells need to survive, the researchers can grow these up in a six-well plate. “The more you expand the cells, the higher probability that there will be selection for cells that are abnormal,” Loring says. “For heart repair, you need on the order of 109 cells, so a lot of the problems that people have in spades, we don’t have. But they’re the ones that really need to be using methods like the ones we’re using to ensure QC.”
Making buckets
It’s not feasible to produce 109 autologous cells for each patient in a large clinical trial, let alone for a commercially viable cell-based therapy.
Thus, issues of mass production come into play. “Can I manufacture cells? Can I manufacture them in a closed system? Can I differentiate them appropriately? Can I select the final cell I get? Can I ship them? Can I cryostore them?,” asks Rao. “These are all things you would need when you’re taking a cell toward therapy.” He says that when he was director of the Center for Regenerative Medicine at the National Institutes of Health (NIH), his lab was the first in the world to produce cGMP-grade iPSC lines.
“Making iPSCs is very easy now—pretty much every lab can do it—it’s become routine. [But] making them so that they will be approved through a regulatory process and making them reliably and reproducibly at high efficiency is really a commercial enterprise,” Rao shares. “It’s been two and a half to three years, and I still get invitations from people asking me to present on how we did it, because only a few groups—mostly commercial entities—have replicated that successfully.”
Edit it
For now, there are no trials involving iPSCs in which the donor cells’ genome has been “corrected” (a form of ex vivo gene therapy).
In the case of Loring’s trial, the patient’s own cells (which have been screened against known defects associated with Parkinson’s disease) are used. Because the disease takes more than 50 years to manifest itself, these “reset” cells are expected to function normally. In other cases, the aim is for allogeneic wild-type cells to correct the deficiency.
Kyba works on a genetically complex form of muscular dystrophy (called FSHD) in which both the allele and the number of 3.3-kilobase tandem repeats are thought to regulate expression of a transcription factor, which ultimately determines whether patients will lose muscle function. “We know what the types of mutations are that can cause the disease, but we don’t have a clear understanding of how those mutations lead to muscle damage—there are a variety of different theories out there,” Kyba says. “And we still don’t understand why it’s only muscle that’s affected.”
Kyba’s lab uses iPSC-derived cells from FSHD patients to study the disease—for example, to determine which of the various myeloid-lineage cells are being affected. The researchers also “model methods of genetic correction, now that a number of genetic-editing tools are widely available,” he says. “I’ve been trying to genetically engineer those FSHD iPSCs to convert them into cells that no longer carry the mutation that caused FSHD.” A long-term goal is to derive and genetically correct the affected cell type and introduce the edited cells back into patients.
The potential of iPSCs to come from anyone and be made into anything is enormous for gene discovery, for mechanism of action studies, for drug screening and for cell therapy. And although it may be a little while before genetically edited iPSC-derived cells are approved for therapy, that just means there’s time to work out the details.
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