cell therapy

Allele Biotech Receives $200,000 Grant to Update Its mRNA Reprogramming Commercial Products and Services

On June 10, 2013 Allele received an SBIR award from the National Institute of Drug Abuse (NIDA/NIH) entitled “Revolutionary Technology for Efficient Derivation of Human iPSCs with Messenger RNA”. The goal of the proposed project is to provide to the biomedical research market an advanced reagent kit and services for highly efficient reprogramming of high quality human induced pluripotent stem cells (iPSCs). At the core of this kit is the Allele team’s recent development transcribed messenger RNA (mRNA). Compared to other reprogramming methods, such as lentivirus, Sendai virus, protein, small molecules or any combinations of these reagents, our new generation of the mRNA method often requires less than half the time while sometimes achieving “bulk conversion” efficiency.

While the Allele reprogramming technology was designed for clinical use as the process is feeder-free, xeno-free, chromosome integration-free, as well as without the need for cell splitting, PI, Dr. Jiwu Wang states, “Our purpose of executing the NIH-funded research it to make our method so easy that any researcher can integrate iPSC into his or her projects.” In addition to the extremely high efficiency, mRNA-generated iPSCs should also be more stable because there are no genetic alterations, more uniform among all clones as there is no clonal event, and ultimately suitable for future autologous cell therapy now that creating iPSCs from patient tissue cells should no longer be the rate-limiting steps.

Allele’s business model is to provide cGMP-grade iPSCs to pharmaceutical companies and perform large scale reprogramming by partnering first with university-affiliated hospitals. Great progress has been made in both directions, which has prompted the initiation of a cGMP unit within Allele’s newly acquired building in San Diego.

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Autologous versus Allogeneic iPSCs in Immune Rejection

The enthusiasm of using autologous induced pluripotent stem cells (iPSCs) for cell replacement therapy was dampened by a publication 2 years ago in Nature (Zhao et al, 2011), which suggested that even syngeneic (genetically identical) iPSCs could still invoke strong immune rejection because, as the authors in Yang Xu’s lab at UCSD explained, the iPSCs overexpress a number of tumor antigens possibly linked to genomic mistakes acquired during reprogramming. Embryonic stem cells (ESCs), on the other hand, did not show similar rejection problems in the same studies, indicating that the immune responses were due to somatic reprogramming.

If proven true, the iPSC-specific immune rejection would have been the biggest hurdle for any iPSC-inspired clinical plans. Naturally, a number of labs performed series of experiments that were aimed at addressing the concerns raised by Zhao et al. This month in Cell Stem Cell, researchers from Ashleigh Boyd’s lab at Boston University demonstrated that autologous (self) or syngeneic iPSCs or their derivatives were not rejected (Guha et al. 2013). These iPSCs behaved essentially the same as ESCs in transplantation settings. When immunogenicity was measured in vitro by monitoring T cell responses in co-culture, no immune response was observed either. In contrast, cells and tissues from allogeneic (genetically different) iPSCs were rejected immediately.

In light of this new publication and an earlier Nature paper (Araki et al. 2013), Kaneko and Yamanaka have commented that autologous iPSCs still seem to have a very good chance of being used in cell replacement therapy, pending, of course, additional research and trial results. In their Preview article in Cell Stem Cell (Kaneko and Yamanaka 2013) two points were particularly emphasized: 1) autologous iPSCs are preferred because of the lack of immune rejection; 2) iPSCs generated with footprint-free reprogramming technologies are preferred because the problems reported by Zhao et al 2011 might be correlated with the use of retroviral vectors (even though they also used episomal plasmid-reprogrammed iPSCs). We strongly support both of these points and believe that they point out the direction of future stem cell therapies.

However, we do not agree with the last statement by Kaneko and Yamanaka in that article stating that as a result of the cost and time required to generate iPSC lines from each patient in GMP facilities, iPSC lines from HLA homologous donors will be the choice going forward to clinical applications. First of all, HLA-matched iPSCs should be closer to allogeneic than to autologous iPSCs. From what we just learned in the last round of debates, the field should certainly go with autologous. Second, generating foot-print free iPSCs may already not be the rate-limiting step, even in cGMP protocols, compared to downstream differentiations that are required using any pluripotent stem cells. We have shown that human fibroblasts can be reprogrammed in a completely feeder-free, xeno-free, passage-free process, using only mRNAs, in just over a week, achieving sometimes “bulk conversion”—converting nearly all cells within a well into iPSCs (Warren et al. 2012). We have drawn up a plan to establish cGMP protocols and to quickly apply autologous, footprint-free iPSCs to clinical programs through partnerships. The field can move at a faster speed, with all due scientific vigor and caution, if the best technology available is chosen for building the foundation.

Zhao, T., Z.N. Zhang, Z. Rong, and Y. Xu, Immunogenicity of induced pluripotent stem cells. Nature, 2011. 474(7350): p. 212-5.

Guha, P., et al., Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell, 2013. 12(4): p. 407-1

Kaneko, S. and S. Yamanaka, To Be Immunogenic, or Not to Be: That’s the iPSC Question. Cell Stem Cell, 2013. 12(4): p. 385-6.

Araki, R., et al., Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature, 2013. 494(7435): p. 100-4.

Warren, L., Y. Ni, J. Wang, and X. Guo, Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Sci Rep, 2012. 2: p. 657.

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Tuesday, April 23rd, 2013 iPSCs and other stem cells No Comments

Path to Better Drugs through Disease-Specific iPSCs

Induced human pluripotent stem cells

The recent finding that pluripotency, the ability to differentiate into all cell types typically associated with embryonic stem cells, can be induced in somatic cells may be the molecular equivalent of the discovery of antibiotics or vaccines in the last century [1].

iPSC-based disease modeling

Recent studies have described the generation of induced pluripotent stem cells (iPSCs) from patients with a full range of genetically inherited or sporadic diseases, and in vitro differentiation of these iPSCs to cell types relevant to the disorder with certain disease features.

Example 1 (out of ~20): Progressive motor neuron loss during differentiation of iPSCs derived from spinal muscular atrophy (SMA) patients, reflecting developmental loss seen in the disease.
Example 2: iPSCs made from RETT syndrome give rise to glutamatergic neurons with fewer synapses than controls, a better treatment was found from a panel of candidates based on this model.
Example 3: Neurons differentiated from iPSCs that have been derived from early or late onset Alzheimer’s disease were shown to display different properties and potential interference points.
The identification of novel pathways or drugs that could prevent disease is the ultimate goal of the iPSC-based disease modeling approach.

Major steps towards efficient iPSC disease modeling

The first hurdle for feasible application of patient-specific disease modeling is to achieve efficient generation of iPSCs from large cohorts of patients quickly and at a low cost while eliminating “clonal variations”. As described in a recent publication [2], the Allele Biotechnology team has shown that human fibroblasts can be converted to stem cells in just over a week, achieving bulk conversion efficiency without any chromosome modifications. The process is also xeno-free and feeder-free, enabling both fundamental scientific research and clinical applications.

The next major advancements required for disease modeling are robust lineage-specific differentiation protocols that provide a large number of desired cells for drug testing and screening. Cardiomyocytes derived from iPSCs have been the best known example of large expansion; other cell types will become available in the near future. Allele Biotechnology has commenced differentiating iPSCs along several lineages using our own iPSCs of superior quality.

With cells of disease-matching tissue types derived from patients’ iPSCs, cell-based assays can be designed and developed using various assay formats. Allele Biotech’s leading capacities in fluorescence and bioluminescence, gene silencing, delivery vehicles and single-domain targeting agents will be of unmatched value to drug discovery partners.

1. Review: Wu, SM and Hochedlinger, K. “Harnessing the potential of induced pluripotent stem cells for regenerative medicine ” 2011, Nature Cell Biology, V13-5, 497-505.
2. Allele Biotech publication: Warren, L., Ni, Y., Wang, J. and Guo, X. “Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA” 2012, Nature’s Scientific Reports, doi:10.1038/srep00657.

For business development contact:
Fax 858-587-6692
6404 Nancy Ridge Drive
San Diego, CA 92121

Related products for academic customers: Non-Integrating iPSC Generation Product Line http://www.allelebiotech.com/non-integrating-ipsc-generation/

New Product of the week: 6F mRNA Reprogramming Premix: $995 for 10 reprogramming!

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