Allele-iPSC News Translate: iPS clinical research encounters a problem. Doctors say “the problem is not caused by iPSCs”

Kobe City Medical Center General Hospital and Riken Research Institute announced and reported to the Japanese government that a patient who had received allogeneic iPSC-derived cells developed an “epi retinal membrane”, which they subsequently removed by operation. Dr. Masayo Takahashi at Riken says “the problem is caused by the transplantation procedure, but not by iPSCs. This will not affect future clinical research that uses iPSCs.”

The laws that govern regenerative medicine in Japan mandates that the deaths and hospitalizations that occur during treatment need to be reported to the government as “serious harmful effects”. This is the first such report involving iPSC clinical research.

The problem occurred to a man in his 70s, who is at the risk of blindness due to “wet age-related macular degeneration”. Last June, he received a transplantation of the solution containing allogeneic iPSC-derived retinal pigment epithelium (RPE) in his left eyes. Last October, the epi retinal membrane and swelling started to develop and the membrane was removed on January 15.

The possibility exists that the solution leaked from the needle hole during the transplantation, and the leaked cells might have formed the membrane. The transplanted cells inside the retina are stable and there has been no decline in his eyesight.

Dr. Takahashi says “although this event qualifies as a serious harmful event, the patient’s condition has not worsened and there has been no rejection of transplanted cells”. Dr. Yasuo Kurimoto, a surgeon who performed the operation, says “the procedure was the problem. We would like to improve the method, in order to make iPSC therapy a common treatment.”

The current clinical trial targets patients with wet age-related macular degeneration and is run by the Kobe City Hospital, Riken, Osaka-University Hospital, and Kyoto-University CiRA (Dr. Shinya Yamanaka). Between last March and October, five patients have received the transplantation.

Original News Credits: https://www.kobe-np.co.jp/news/iryou/201801/0010902012.shtml

Tags: AMD, eye disease, iPSC, Japan iPSC, RPE, stem cells, Takanishi, Yamanaka

Allele Publication Explains cGMP Generation of Induced Pluripotent Stem Cells

The discovery that adult somatic cells can be reprogrammed to pluripotent stem cells has given the biomedical community a powerful platform for personalized medicine. However, the translation of cell therapies from bench to bedside holds a significant challenge. Realizing the clinical potential for stem cells requires their production under current Good Manufacturing Practice (cGMP) regulations enforced by the FDA. A new protocol (http://onlinelibrary.wiley.com/doi/10.1002/cpsc.18/abstract) published by scientists at Allele and detailed in this quarter’s issue of Current Protocols in Stem Cell Biology, reveals key conditions required for converting adult fibroblasts to induced pluripotent stem cells (iPSCs) under cGMP regulations.¹

The patent-pending protocol is an update to a previous protocol that describes how to reprogram fibroblasts to iPSCs using mRNA. “The system of using mRNA to reprogram fibroblasts presents itself as a very favorable candidate for generating iPSCs for cell therapy” according to the senior author of the paper and CEO of Allele, Dr. Jiwu Wang, “our company is committed to developing stem cell based therapies using this protocol and through the establishment of our own stem cell GMP facilities here in California”. mRNA transfection is “footprint free”, meaning no insertions or alterations have been made to the genome. Transfection of mRNA is also “cleanup free,” because mRNA transcripts are supplied to the cells in the culture medium only for the time required to induce pluripotency. Furthermore, genomic analyses of iPSCs reprogrammed using mRNA indicate that this method of conversion is unlikely to introduce problematic mutations.²

The new version of the protocol describes reprogramming technology that utilizes all cGMP-certified reagents and vessels, meaning that every material is manufactured under guidelines that allow for ancillary use in manufacturing processes related to cell therapy. All materials described in the protocol – from cell medium and components to the coating for tissue culture plates – were meticulously evaluated at every step of generating and storing iPSCs. For truly cGMP produced cell lines, all processes should take place in certified cleanrooms with qualified equipment and thoroughly trained operators.

Establishing a cGMP process for any product intended for human use is a daunting undertaking. Unlike drugs and small-molecule pharmaceuticals, stem cells are living entities whose production cannot be chemically synthesized. Therefore, special considerations must be made – particularly for making individual cell lines – to help assure the highest safety and quality of downstream stem cell products. Adhering to cGMP regulations infuses high quality into the design and manufacturing process at every step. Through rigorous testing, researchers at Allele have identified critical parameters for generating iPSCs from fibroblasts that are cGMP-compliant, and are optimistic that the methods described in this recent publication will serve as a launch pad for the development of future cell products and therapies.

1. Ni Y, Zhao Y, Warren L, Higginbotham J, Wang J. cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA. Current Protocols in Stem Cell Biology,2016; 39:4A.6.1-4A.6.25.
2. Bhutani K, Nazor KL, Williams R, et al. Whole-genome mutational burden analysis of three pluripotency induction methods. Nature communications. 2016;7:10536.

Allele GMP CRO and Cell Therapy

Tags: Allele Therapeutics, CA company, CIRM, GMP, iPSC, stem cell therapy

cGMP Compliance: What Does It Mean for Your Cell Lines?

As the promise for cell-based therapy grows, the interest in making clinically relevant cell lines has skyrocketed for industrial and academic researchers alike. For translation into human therapies, cell-based products must be made following current Good Manufacturing Practice (cGMP). Many groups have already claimed to generate cell lines that are “cGMP-compliant,” “cGMP-ready,” or “certifiable under cGMP.” But what does it take to be truly cGMP-compliant, and what practices can you introduce in your lab to comply with cGMP standards?

A common misconception in the United States is that a facility is granted a ‘cGMP license’ from the government to manufacture cGMP-grade products. Rather, the Food and Drug Administration (FDA) evaluates the manufacturing process for each product to determine if it is compliant with cGMP standards. The primary concern when it comes to deriving cell-based products for therapies is making sure that the product is derived in a safe and reproducible manner. To ensure maximum quality assurance, researchers should

• choose reliable, xenogeneic-free raw materials,
• establish and monitor a clean environment,
• qualify all equipment and software,
• remove variation in laboratory procedures by creating detailed Standard Operating Procedures (SOPs) and by providing rigid process validation at each step.

Nevertheless, even establishing robust quality assurance does not imply that the process is scalable for commercial production. In the world of biologics, “the product is the process.” A requisite step to ensure a smooth transition to cGMP practice is to ensure that the process of manufacturing is not altered due to changes in production scale. For example, depending on the therapy, millions or billions of cells may be required for a single patient. Therefore, it is in the best interest of the researchers to develop a scalable method at the beginning to avoid revamping the entire process (e.g., changing from adherent cells to suspension). Along these lines, the quality control (QC) requirements of cell-based products should be carefully considered and not have to include difficult-to-assay tests. For example, some cell lines have been qualified as cGMP-compliant upon conversion from research-grade conditions to cGMP quality standards. Rigorous tests were performed on the converted lines to ensure that the cells were free of contamination. Even though strict measures were carried out to ensure cGMP compliancy, deriving cell lines in this manner makes scalability and reproducibility a challenge. Ideally, the entire process of deriving cell products for clinical use should be performed under cGMP conditions: from the acquisition of human tissue to the manufacturing, testing, and storage of derivative cell products.

Another important consideration when instituting cGMP-compliance is documentation. Each process must be described with rigorous SOPs, the training of individual manufacturing operators must be well-documented, and the entire established process must be validated and well noted. Failure to document—in the eyes of the FDA—is often equated with failure to perform the underlying activity. It is equally important to remain ‘current.’ The FDA expects manufacturing processes to stay up-to-date with current regulations, even as policies change.

For an academic lab, closely aligning with cGMP standards can ensure that the resulting cell lines are comparable to other truly cGMP-produced products used during clinical trials. It is in the best interest of academic researchers to establish rigorous SOPs and use qualified reagents and equipment, even if it is not possible to carry out all steps in a certified cleanroom. Whenever possible, it is advisable to acquire truly cGMP cell lines from appropriate sources for preclinical projects; if prohibited by costs or other reasons, it is recommended to use a protocol that is as close to cGMP as possible.

Tags: Allele Biotech, cell based products, cGMP, footprint free, iPSC, stem cells

Allele Biotechnology & Pharmaceuticals Forming Cell Banking Business for Personalized Medicine

Allele to Generate Human iPS Cells under Good Manufacturing Practice for Private Individuals for Potential Therapeutic Use and Future diagnostics; Cell Banking Scientific Advisory Board formed

October 06, 2015 11:25 AM Eastern Daylight Time
SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals, Inc. (“Allele”), a leader in the development of specialized cells for regenerative medicine and pharmaceutical drug discovery, today announced plans to form a commercial business for the banking of human induced pluripotent stem cells (iPSCs) by private individuals.

Allele is pleased to have Drs. Mahendra Rao and Joseph Paulauskis as the first members of its Scientific Advisory Board (SAB) for iPSC banking and cGMP production. Dr. Rao is a world-renowned scientist in the fields of stem cells and medicine, having served as the VP of Regenerative Medicine at Invitrogen, founding Director of the NIH Center for Regenerative Medicine, Chair of the Biological Response Modifiers Committee (BRMAC, now CTAGT) of the FDA, and he is currently Chief Strategy Officer at Q Therapeutics. Dr. Paulauskis is the Chief Operating Officer of Paradigm Dx as well as the Vice-President of Research and Biobanking for the International Genomics Consortium, and has previously held senior positions in pharmacogenomics at Pfizer. Allele plans to make additional appointments to the SAB as individuals with world-class experience and expertise are identified.

Human iPSCs are cells that can be grown to virtually infinite numbers and can become any cell in the human body, features which hold great promise for therapies that can alleviate or cure human disease. Allele’s business model recognizes that an individual can have their own cells ready for future therapeutic use via the generation and storage of iPSCs from that individual’s skin cells. While this is similar to what is done with newborn cord blood, iPSCs can be generated from humans of any age. The banking of human iPSCs for potential future therapeutic use is a relatively new industry with unprecedented potential, and Allele is benefitting from expert opinions internally and from current and future SAB members, ensuring a solid scientific and ethical foundation for this business.

In addition to serving its customers, Allele’s iPSC bank will be an unparalleled resource for biomedical research. Proper consent and privacy guardianship will allow thousands of iPSC lines with accompanying sequence database and health information to be made available from the bank to scientists and clinicians. Currently, iPSC banks are funded by government agencies at multi-million dollar costs per project; Allele’s model does not rely on tax dollars and provides potentially a larger bank of iPSCs of higher quality to aid research and treatment efforts. To this end, NIH Director Dr. Francis Collins recently announced the implementation of the Precision Medicine Initiative (PMI), the goal of which is for health care professionals to have the resources to take into account individual differences in genes, environments, and lifestyles that contribute to disease when providing treatments in the new era.

“We are happy to have the guidance from world leading experts in stem cells, biobanking, and cell therapy fields such as Drs. Rao and Paulauskis”, said Jiwu Wang Ph.D., President and CEO of Allele. “We believe that setting the bar high will be ultimately beneficial to future customers, fellow researchers, industry partners, and regulatory agencies alike. We are happy to see the recent release by the International Society for Stem Cell Research of a draft of ‘Guidelines for the Clinical Translation of Stem Cells’, whose principals we plan to follow closely. We also intend to obtain certification by the cord blood banking association AABB, if possible, and abide by other regulatory rules as they become public, such as the “Stem Cell Clinical Research Management (tentative)” by the Health Commission of China, if and when we move to operate under that jurisdiction”.

Towards the establishment of this business effort, Allele has recently purchased an 18,000 square-foot facility, located near its headquarters in San Diego, California. This new facility will be the center of cGMP-production of human iPSCs using Allele’s proprietary synthetic mRNA platform, a technology that generates cells with neither the random integration of foreign DNA nor the use of viruses or virus-based elements, drawbacks common to other technologies for making hiPSCs; thus, the “footprint-free” cells generated by Allele’s synthetic mRNA platform are optimally suited for therapeutic use, and Allele’s technology has been licensed for clinical trials by companies such as Ocata Therapeutics (formerly ACT). This effort received strong support from Yuan Capital and Yifang Ventures.

About Allele Biotechnology & Pharmaceuticals, Inc.

Allele Biotechnology and Pharmaceuticals, Inc. is a private, San Diego-based company that explores the mechanisms of biological processes to develop technologies and products for biomedical researchers. Allele utilizes proprietary non-integrating cellular reprogramming methods to generate human and non-human primate iPS cells, GMP-grade human iPS cells and their derivatives, and differentiated cell types. With additional expertise in genome modification and cell-based sensors/reporters, Allele provides advanced cell-based assays for drug discovery. Allele also has developed a wide variety of reagents including superior fluorescent proteins and camelid antibodies. The company has also been a leader in the RNAi field with its patents in Pol III promoter-driven siRNA, shRNA, and miRNA.

Contacts
Allele Biotechnology & Pharmaceuticals, Inc.
Matthew A. Singer, Ph. D.
Director of Business Development and Strategic Alliances
+1 858-587-6645, ext. 1
mattsinger@allelebiotech.com
www.allelebiotech.com

Tags: Allele iPSC, cell bank, cGMP, CIRM, GMP, iPSC, Mahendra Rao, NIH, personalized medicine, precision medicine, SAB, stem cell therapy

Allele’s SBIR Grant to Develop All-RNA CRISPR

Precise engineering of the genomes of mammalian cells enabled biological and medical applications researchers had dreamed of for decades. Recent developments in the stem cell field have created even more excitement for genetically modifying genomes because it enables delivering more beneficial stem cell-derived therapeutic cells to patients [1]. For instance, by correcting a gene mutation known to be critical to Parkinson’s disease, LRRK2 G2019S, in patient-specific iPSCs (induced pluripotent stem cells), it appeared possible to rescue neurodegenerative phenotypes [2].

Significant amount of fund and energy had been invested in technologies such as ZFN and TALEN, however, judging from the explosion of publications and business activities in just about 2 years since the illustration of its mechanism (just today, Jan 8th, 2015, Novartis announced CRISPR collaborations with Intellia, Caribou, applying it in CAR T cell and HSCs), the CRISPR/cas system is the rising star. This system uses a guide RNA to direct the traffic of a single nuclease towards different targets on a chromosome to alter DNA sequence through cutting. The nuclease, cas9, can be mutated from a double-stranded DNA endonuclease to a single-strand cutter or a non-cutting block, or further fused to various functional domains such as a transcription activation domain. This system can also be used to edit RNA molecules.

A weak spot on the sharp blade of CRISPR is, like any methods for creating loss-of-function effects (RNAi if you remember), the potential of off-target effects. While they can never be completely avoided, with the ever growing popularity of deep sequencing, at least we can know all unintended changes on the edited genome. Almost a perfect storm! As an interesting side story, when we at Allele Biotech first saw the paper in Science describing the CIRPSR/cas system [3], we immediately wrote an SBIR grant application for applying the bacterial system to mammalian cells. The first round of review in December 2012 concluded that it would not work due to eukaryotes’ compact chromatin structures. Of course, the flurry of publication in early 2013, while our application was being resubmitted, proved otherwise. The good news is, Allele Biotech still received an SBIR grant from NIGMS in 2014. Unlike most of the genome editing platforms known in the literature, our goal was to build an all-RNA CRISPR/cas system, thereby with higher potency, less off-target effects, and, as a footprint-free platform, more suitable for therapeutic applications. This system will be combined with our strengths in iPSC and stem cell differentiation, fluorescent protein markers, and deep sequencing based bioinformatics to improve cell therapy and cell based assays.

1 Urnov, F.D., et al., Genome editing with engineered zinc finger nucleases. Nat Rev Genet, 2010. 11(9): p. 636-46.
2 Reinhardt, P., et al., Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression. Cell Stem Cell, 2013. 12(3): p. 354-67.
3 Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.

Tags: Caribou, cas9, CRISPR, Intellia, iPSC, Novartis, SBIR, TALEN, ZFN