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:
iPS@allelebiotech.com
858-587-6645
Fax 858-587-6692
www.allelebiotech.com
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!

Tags: cell therapy, disease model, drug screening, iPSC, Klf, Lin28, M3O, mRNA reprogramming, Nanog, Oct, Sox

DNA Repair Pathway Factors in Cell-Based Screening for Restoring Patients’ Sensitivity to Cancer Therapies

Cancers undergoing therapies may develop resistance to treatment. Many current cancer treatments, such as cisplatin, function by creating DNA damage, particularly to fast-dividing cells, i.e., most cancer cells. These treatments may be rendered ineffective by DNA-damage response pathways. Cancer resistance to therapies may come from increased activity in nonhomologous end joining, decreased functions of mismatch repair, or reactivation of the Fanconi anemia (FA)/BRCA DNA-damage response pathway, etc. Ironically the loss of function of some of these DNA-damage repair factors may have partially caused the cancer formation in the first place. Regaining their functions in cancer cells possibly contribute to drug resistance. Molecules that disrupt FA/BRCA pathway or other DNA-damage responses could be used to help restore therapy sensitivity.

Like many proteins that function in DNA-damage repair complexes, FANCD2, a member of the FA pathway factor group, is targeted towards chromatin following damage to DNA in a process called foci formation. There have been recent studies that monitored the foci formation of GFP-FANCD2 in small molecule library screening and identified inhibitors to FANCD2 as candidates for a cancer therapy sensitizer. The assays can be improved in a number of ways. There are fluorescent proteins (FPs) that are much brighter than EGFP for increased sensitivity. For instance, the monomeric green FP mWasabi is about 2-3 fold brighter than EGFP, with narrower emission peak, and is more stable under acidic environment. The newly developed lancelet YFP (LanYFP, developed/introduced by Allele Biotech) is astonishingly 10 times brighter than EGFP. Since it has a longer excitation and emission wavelength, it should inherently have a better signal to noise/background ratio compared to EGFP because cells autofluoresce less in long wavelengths. The improved brightness would also help in this respect. The fold difference between foci and LanYFP background will be the same as EGFP, but the contrast will still probably be better because of less autofluorescent background and significantly higher fluorescence reading in foci.

Other factors that may be used as a screening target when fused to effective FPs may probably include:

1) Homologous recombination (HR)
a. End Resection
MRN complex (MRE11, RAD50, NBS1)
CtIP, RPA, ATM, ATR, Exo1, BLM, RMI1, TopIIIa, DNA2, BRCA1
b. Synapsis
RAD51, BRCA2, PALB2, RAD51B, RAD51C, RAD51D, RAD51AP1, XRCC2, XRCC3, RAD54, RAD54B
c. DNA synthesis
DNA polymerase delta, PCNA

2) Nonhomologous End Joining (NHEJ)
Ku70/Ku80, DNA-PK, Ligase IV, XRCC4, XLF

3) Fanconi Anemia Pathway
FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100, FANCM, MHF, FAAP24, FANCD2, FANCI, FAN1, FANCN, FANCJ, FANCM

New Product of the Week 101110-101710:

Puromycin-resistant versions of lanRFP (red fluorescent protein from lancelet) for mammalian expression, just became available this week. ABP-FP-RCNCS1P, ABP-FP-RNNCS1P

Promotion of the Week 101110-101710:

30% off the brightest ever lancelet YFP, ABP-FP-YPNCS10, $349 reduced to $244.3 for this week’s orders only.

Tags: cancer therapy, cancer treatment, DNA-damage, DNA-repair, drug resistance, drug screening, fluorescent protein, NIH grant, RNAi

Allele Custom Services for Drug Screening Companies

Many target discovery and validation programs can benefit from RNA interference, fluorescent proteins, stem cells, and viral delivery systems. However, applications of these technologies require special reagents and laboratory know-how. Even when available, many generic reagent kits are not tailored for your particular needs in screening or validation.

At Allele, we accelerate your discovery efforts with custom RNAi screening, fluorescence based assays, and cell model development services.

1) Our RNAi platform, based on our patented shRNA/miRNA technologies, use DNA linear template, plasmid, lentivirus, retrovirus, or baculovirus vectors that prompt cells to endogenously express RNAi. As a result, our screens offer advantages over synthetic siRNAs:
• Higher levels of consistency
• Greater delivery and gene silencing efficiencies
• Accessibility to difficult-to-transfect cells, including primary cells
• Potential for inducible RNAi expression
• More persistent silencing with shRNA under Allele’s own IP–you may not need to license siRNA patents!

2) Fluorescent proteins (FPs), which can span the entire visual spectrum, have become some of the most widely used genetically encoded tags. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Allele Biotech is one of a few companies that develop and improve FPs through fundamental research. We have so far achieved:
• The brightest cyan and green FPs, true monomers for minimum artifact or cytotoxicity
• The brightest yellow and red FPs from lancelet, only FPs from vertebrate
• mTFP1 as the best FRET donor by 3 independent reports
• Photoconvertible FPs for super imaging or kinetic labeling
• Delivery on plasmid, retrovirus, lentivirus, baculovirus vectors

3) As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors. These induced stem cells (iPSCs) provide unprecedented resources of cells of different differentiation stages for functional testing and drug screening. Allele Biotech develops and provides state-of-the-art reagents in convenient forms for iPSC production
• iPS factors carried on lentivirus, retrovirus, baculovirus for different cell types
• Availability in combination with fluorescent proteins under own IP, and drug resistant genes
• 4-in-1 or 2-in-1 effective use of iPS factors on one viral vector
• Feeder cells of human origin expressing factors essential for stem cell culturing

4) Introduction of protein factors, miRNA, promoter-reporter, and virtually any other genetic element of interest via the most efficient viral packaging systems.
• Introducing protein-FP fusion, promoter-FP reporter, photoactivatable factors for cell-based assays
• Introducing critical factors for cell immortalization
• Episomal or integrated expression using baculoviral vectors
• High throughput, systematic expression of whole class of molecules in any type of cell
• High titer viral packaging at low cost for delivery to animal tissues

In addition, the Allele team can provide custom-designed assays that can be used for assaying enzyme activities in almost any pathway, such as the EGF pathway, TNF response/apoptosis pathway, nuclear receptors, etc. We utilize technically advanced methods to provide our partners with advantages over alternative methods or other services.

New Product of the Week 06-28-10 to 07-03-10: Eco-friendly mammalian tissue culture plates, 40% less plastic to the environment, 40% less cost to your budget, contact our sales rep today for quotes and details.

Promotion of the Week 06-28-10 to 07-03-10: Oct3/4 iPS lentivirus with RFP as marker, new to the market, this week only all kits containing Oct3/4-RFP same price as the original, non-RFP versions, save ~$50!

Tags: cell assays, cell based assays, cell culture, drug screening, FP assays, FRET, iPS cells, kinase assasy, pathway, RNAi

Lentiviruses expressing constitutively active or dominant negative versions of genes in signaling pathways

NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) refers to a protein complex functional in signaling pathways, particularly in response to stress stimuli. There are two signaling pathways leading to the activation of NF-kB signaling, known as the canonical (or classical) pathway, the non-canonical (or alternative) pathway.

In the canonical NF-kB pathway, NF-kB dimers such as p50/RelA are maintained in the cytoplasm by interaction with an independent Inhibitor of NF-kB (IkB) molecule. When the upstream signaling is active, an IkBa kinase (IKK) complex consisting of catalytic kinase subunits IKKa and/or IKKb and the scaffold protein NEMO will be recruited to the cytoplasmic adaptor of certain cell surface receptor and stay activated. Activation of IKK complex will consequently phosphorylate the IkB at two serine residues, which induce the proteasomal degradation of IkB. Released from IkB, NF-kB dimers then translocate into the nucleus and bind with a consensus sequence (GGGACTTTCC) of various genes and thereby activates their transcription. In a negative feedback loop, NF-kB activation also leads to the expression of the IkB gene, which subsequently sequesters NF-kB subunits and terminates transcriptional activity unless a constitutive activation signal is present.

The non-canonical pathway is mainly for activation of p100/RelB complexes during B-and T-cell development, where NF-kB was first discovered. Different from the canonical pathway, 1) only certain receptor signals (e.g., Lymphotoxin B, B-cell activating factor, CD40) can activate this pathway, 2) it proceeds through an IKK complex that contains two IKKa subunits (but not NEMO) and 3) receptor binding leads to activation of the NF-kB-inducing kinase NIK, which phosphorylates and activates an IKKa complex, the latter in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal IkB domain of p100, leading to its partial proteolysis and liberation of the p52/RelB complex.

Other distinct NF-kB pathways undoubtedly exist. For example, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction with the IkB-like co-activator Bcl-3 (or IkBz). How these are regulated is not known.

There are many ways of applying reagents that can manipulate the NF-kB system for drug screening as well as basic research. For this reason, Allele Biotech is introducing a set of lentiviruses as listed below. The way this product group is operated is that the Allele will publish the design and specifics of the products, but will produce it upon the first order. The person who places the first order will need to wait for 3-5 weeks for receiving the products, but will receive a 40% discount and an opportunity to provide input to the product design. The platform for lentiviral products is based on our field-leading high titer lentivirus packaging capabilities, and you can rest assured that high quality lentivirus will be produced at a pace much faster and lower price than if you were to make them by your own lab or find them from anywhere else. Allele Biotech will introduce more such products in the fields of miRNA (to provide all ~800 human miRNA and their anti-miRNA silencers on virus), cell differentiation lineage-specific promoter-FP reporters, etc. We now call these products the Lead Products. Cost effectiveness is just the beginning. Imagine how our ingenuity and innovation can pave the way for your research.

1) HiTiter IkBa Expression Lentiviral Particles: can be used in functional studies of IkBa’s roles in NF-kB signaling. Overexpression of IkBa will eliminate the low-level activation by factors in the cell culture medium or other fluctuations to ensure that any change in NF-kB signaling is caused by the stimulus being tested.
2) HiTiter Dominant Negative IkBa Expression Lentiviral Particles: Dominant Negative IkBa has serine-to-alanine changes at residues 32 and 36. It can be used to repress the expression of endogenous IkBa or block NFkB signaling in certain cell lines.
3) HiTiter IkBa-RFP Fusion Expression Lentiviral Particles: This is a reporter suitable for the studying proteasomal degradation of IkBa after phosphorylation.

4) HiTiter IKKa Expression Lentiviral Particles: can be used in functional research for the role of IKKa in NF-kB signaling.
5) HiTiter Constitutively Active IKKa Expression Lentiviral Particles: Constitutively active IKKa has serine-to-glutamate mutations at residues 176 and 180.
6) HiTiter Dominant Negative IKKa Expression Lentiviral Particles: Dominant negative IKKa is mutated by serine-to-alanine at residues 176 and 180.
7) HiTiter IKKb Expression Lentiviral Particles: can be used in functional research for the role of IKKb in NF-kB signaling.
8) HiTiter Constitutively Active IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-glutamate at residues 177 and 181.
9) HiTiter Dominant Negative IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-alanine at residues 177 and 181.
10) HiTiter pNFkB-Luciferase Lentiviral Particles: a cis-reporter plasmid containing the luciferase reporter cDNA linked to five repeats of an NF-kB binding site.
11) HiTiter pNFkB-GFP Lentiviral Particles: a cis-reporter plasmid containing the GFP reporter gene linked to five repeats of an NF-kB binding site.

New Product of the Week 05-23-10 to 05-31-10:

Cre recombinase fused to mWasabi GFP carried on lentivirus, ABP-RP-Cre2AGL, or ABP-RP-Cre2AGS

Promotion of the week 05-23-10 to 05-31-10:

actually 3 promotions this week, first announced to Allele fans and friends on Facebook: 10% off the brand-new RFP-Trap ACT-CM-RFA0050 for co-IP with mCherry, mPlum, mOrange…Order by Monday to qualify for discount! Free sample for FAM-amidite for oligo cores at universities that make qPCR and other probes. Free sample of high attachment tissue culture plates that use 40% less plastics (made in Canada)

Tags: Bcl-3, drug screening, IkBa, IkBz, Ikka, Ikkb, lentivirus, NEMO, NF-kB, p100, p50/RelA, p52/RelB, signaling pathways, stimuli, stimulus