Viruses and cells

mRNA Delivery And the Next Wave of Regenerative Medicine

Published online by Nature Biotechnology, researchers from Ken Chien’s lab at Harvard and other coauthors showed that modified mRNA of VEGF-A injected intramyocardially resulted in the expansion and directed differentiation of endogenous heart progenitors. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients by directing epicardial progenitor cells toward cardiovascular cell types. This publication appears to be the first example of using mRNA as a delivery platform for cell fate-related therapy. AstraZeneca recently invested $240 million on mRNA-related delivery via Moderna, a company with roots within the Harvard stem cell group.

The drastically increased efficacy of using the mRNA platform was accredited to the pulse-like kinetics of mRNA expression profile. It was explained by the fact that native paracrine signals are often transient and precisely regulated in time and space, therefore the pulse-like expression profile of modRNA might be well suited to delivering paracrine-factor signals. Transfected mRNA molecules do not need to penetrate the nuclear membrane, which greatly enhances the efficiency of protein expression on a per transfected molecule over DNA. mRNAs turn over in a much faster pace than plasmid-mediated transgene expression. This is beneficial to many cell fate decisions as exemplified by this recent publication.

Allele Biotech’s reprogramming technologies, licensed by some of the leading stem cell therapy companies, are built around the mRNA platform. We chose mRNA as our core technology to not only change cell fate, but also direct differentiation. We know this platform is the future for cell fate manipulation because we have seen how robustly mRNA expression made the day-and-night difference in gene expression when compared to plasmid DNA (episomal or not), retrovirus, lentivirus, baculo virus, or even transfected proteins. We could convert human fibroblasts into iPSCs, in bulk, in as short as one week with no more effort than changing mRNA complex-containing medium.

Another recent development in iPSC research is in situ reprogramming. Abad et al. generated mice carrying a Tet-inducible cassette of the four cell-reprogramming factors. They then added feed doxycycline to the animals. After several weeks, teratomas appeared in various tissues, indicating that in situ reprogramming had occurred. The iPSCs created this way did not appear to have much advantage over in vitro produced iPSCs other than they are totipotent (helpful if you are studying placenta). Nevertheless, the concept of changing cell fate in situ as dramatically as complete reprogramming is an important leap of faith. As for the next big step, it is easy to see that mRNAs are well suited for in situ reprogramming, as well as transdifferentiation, and more complex gene delivery than the above mentioned VEGF-A alone in heart treatment.

References:
Zangi et al. Nature Biotechnology, http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt.2682.html
Abad et al. Nature, http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature12586.html

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Thursday, September 12th, 2013 iPSCs and other stem cells, Viruses and cells No Comments

Genome Modification—a Practical Approach

The ability to modify genomes has always been fervidly sought after by molecular, developmental biologists and geneticists as it would provide them with the means for finding out what a particular piece of the genome may do in the biological process they are studying. The discovery of naturally existing P-element helped a generation of Drosophila geneticists and made the fruit fly a prime model system for gene function studies in the 80’s and 90’s. But P-elements inserted at uncontrolled sites, making it essentially a gene transfer vehicle without much control. The introduction of prokaryotic recombination systems, e.g. LoxP and Cre, provided researchers with tools to obtain more control of the inserted genes in a host chromosome during a biological process such as development. Transposons like Sleeping Beauty, Piggybac, or Tol2 made similar experiments possible in mammalian cells.

Still, the randomness of transposon-type elements’ insertion, much like retrovirus or lentivirus, could cause trouble if they land in an undesirable spot. Methods of inserting transgenes only in well-known, harmless, and transcriptionally active regions, so called “safe harbors”, were subjects of interest of researchers and NIH grant topics in the past couple of years under “directed genome editing”. Gene knock-out or knock-in can be achieved through vector-mediated homologous recombination such as the rAAV genome engineering system and the “TARGATT” system, which are commercially available as kits or services.

However, instead of inserting an exogenous gene, it is often highly desirable to modify an endogenous genome sequence, which requires the modification apparatus to first recognize the target sequence. ZFN and TALEN both recognize DNA targets through specific nucleotide binding protein domains, with TALEN having more flexibility if assembled in a “Lego”-like format because each domain can specifically recognize a “C”, “G”, “T”, or “A” base. The description of using CRISPR/cas system in a recent burst of publications opened up new ways of binding to specific DNA sequences and nicking or severing the dsDNA. This system does not require engineeredDNA binding domain assembly; instead, it uses a guide RNA to find the target DNA sequence to direct endonuclease, in a sense quite like RNAi. However, the enthusiasm about CRISPR/cas was somewhat dampened by a report last month in Nature Biotechnology that reported off-target effects of CRISPR/cas was much higher than ZFN and TALEN. Particularly, if mismatches are located in the 5’ portion of the guide RNA targeting sequence, they can be well tolerated up to 3 or 4, even 5 mismatches. Unfortunately this is also similar to the tolerance of the RNAi matching region outside the core 12-base region. The difference is: for RNAi, the off-target damage is temporary and ignorable if the extent is insignificant compared to the effects on the intended target while for CRISPR/cas, an off-target cut on the chromosome is permanent.

On the positive side, in an even more recent publication in Nature Methods, mutant strains of C. elegans were obtained using the CRISPR/cas system and no evidence was found for off-target changes, at least not in an overwhelming fashion. Much value of the estimates of off-target effects relates to the methods used for analysis. Currently, most of the studies looked at potential off-target sides by searching for partial matches. In the future, whole genome sequencing will be increasingly required for submitting such publications.

On a practical note, if you intend to take a dive and try to use any one of these methods, your number one problem will be that none of the methods will result in 100% modification even if you can ignore the off-target problems for now. Therefore, many of our customers ask about a screening strategy. One could use traditional drug selection and fluorescent protein (FP)-based sorting, but these can only help you find cells that are successfully transfected with the ZFN, TALEN, or CRISPR/cas expressing DNA molecules, not necessarily having the genome modification result. We have formulated the idea of inserting the target site into an FP-bearing plasmid as a surrogate target cutting indicator, and use another FP to track transfection of the TALEN plasmid. Nonetheless, in the end, PCR-amplifying the target region of the chromosome and doing either an enzymatic mismatch detection assay (e.g. T7 endonuclease) or sequencing is the only way to know for sure whether genome editing has occurred.

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Wednesday, July 10th, 2013 Synthetic biology, Viruses and cells 1 Comment

Using Insect Cells For Making Mammalian Proteins

Recombinant protein expression is a major part of biological research. In theory, once the genetic code of a protein is known from cDNA analysis or whole genome sequencing, any polypeptide of interest, existing in nature or perceived, can be artificially produced. Bacteria cells are commonly used to express a variety of proteins because they are more convenient and less costly than other systems. However, a significant percentage of proteins naturally expressed in mammalian cells are not soluble or cannot be easily produced in bacteria such as E. coli. Like bacteria, yeasts are also easy to culture and manipulate, however, although they are eukaryotes, they are not capable of adding “mammalian-like” post-translation modifications (PTM). Insect cells can be used effectively for producing large quantities of mammalian proteins rather easily through baculovirus such as Allele´s Sapphire system. PTM in insect cells is not exactly the same as in mammalian cells, e.g. different glycosylation patterns, but is a lot closer than yeasts. Mammalian cells are used for proteins that require appropriate PTM or are not soluble in other systems through either transient transfection or stable cell line establishment.

For protein expression in insect cells, a number of factors need to be taken into consideration:

1) Genomic DNA for creating baculovirus stocks that will ensure a high percentage of recombinant virus (to avoid wild-type, non-producing virus)
2) Transfer plasmid for cloning the protein-encoding cDNA for easy cloning and appropriate co-expression of helper or marker proteins (such as through insect IRES)
3) Cell lines that have the highest expression levels of a particular protein, sometimes a number of cell lines need to be screened
4) Cell medium, because insect cell medium may contain high levels of ions that can interfere with affinity tag-based purification, one needs to find the most appropriate medium for protein expression
5) Secreted vs nonsecreted proteins. Insect cells need to have their own secretion signal (and translation signal, IRES, polyadinylation, etc.)

More reading…http://www.allelebiotech.com/protein-expression-in-insect-cells/

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Wednesday, February 29th, 2012 Viruses and cells No Comments

Making Transfection-Grade mRNA by IVT (In Vitro Transcription)

RNases are an often feared in molecular biology labs because of their high stability and ominous presence in virtually all living systems. Consequently, people who work with RNA are trained to exercise extreme caution to avoid RNA degradation: change gloves often because human hands ooze RNases; use only sterilized labware as microbes may be sources of RNases; for surfaces that can’t be autoclaved, use sprays like “RNase Zap” (SDS- or guanidine-containing solutions). Such cautionary steps are especially necessary when dealing with low abundance RNA samples.

RNAs can be produced by in vitro transcription (IVT), a simple reaction requiring only a DNA template (double-stranded or even single-stranded DNA as long as the promoter region is double-stranded), RNA polymerase (from T7, SP6, or T3 phage), NTPs, and a reaction buffer that provides appropriate salt and pH. Standard NTPs may be replaced with modified ones to either increase stability or to reduce immune-response when transfected into cultured cells. Additionally, a 5’ cap structure may be added during IVT for further stabilizing mRNAs inside the cells post transfection. Using a commercially assembled kit, one can routinely produce 40-50 µg of mRNA from 1 µg of DNA template in a single 20-50 µl reaction.

At such high concentrations, IVT mRNAs are not nearly as sensitive to RNase-mediated degradation as low-abundance samples. The mRNA can be easily observed on agarose gels that are regularly used for DNA, and their integrity can be monitored after transcription or storage. In most cases one distinct band of mRNA from an IVT reaction is obtained as long as a clean DNA template is used. Preparing a good, uniform IVT template is critical to prevent aberrant products. By using high quality templates, IVT mRNA produced in your own lab are often higher in quality than mRNAs purchased from current commercial sources (Figure in Blog shows mRNAs generated by IVT for R-iPSC). Sometimes there are minor bands created during IVT, but they normally do not interfere with the intended uses of the mRNA, and can be purified away with a purification kit (by using a discriminating purification scheme such as Allele Biotech’s Surface Bind RNA Purification, smaller species can be specifically removed, a separate topic for another blog).

Once produced, mRNAs can be stored at -20C for months, or -80C nearly indefinitely.

IVT mRNA for iPSC generation

mRNAs generated by IVT for R-iPSC

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Big Potential in Using Protozoans for Producing Mammalian Proteins

Recombinant protein expression is critical for functionally studying proteins, preparing antigens, providing tissue culture growth supplement, and producing certain therapeutic compounds. Like many molecular biology labs, we have used several heterologous protein expression systems over the last decade including E. coli, yeasts, insect cells and mammalian cells from various species. It is widely accepted that these systems present increasing functional relevance from bacteria to mammalian cells, with accompanying increase in difficulty and cost. The benefits of using cells from higher species are often reflected in post-translational modifications (PTMs), such as glycosylation, phosphorylation, etc.

There is yet another system that could be easy to handle while maintaining mammalian-like PTMs–parasitic protozoan Leishmania tarentolae. L. tarenolae is a unicellular organism, its host is lizard. Even though it’s a vertebrate parasite, this species poses no risk to humans. Amazingly, L. tarenolae individuals can be grown on agar plates for clonal selection or in simple liquid media like E. coli. Their optimal growth temperature is 27C, and they do not require shaking; thus they are suitable for growth in insect cell incubators or even at room temperature. The most important advantage of this system is that oligosaccharide structures of proteins produced in this organism resemble those of mammalian cells much more closely than even insect cells, i. e. the N-glycosylation profile can be basically identical to a biantennary fully galactosylated Man3GlcNAc2core-a-1,6-fucosylated structure found in mammalian cells.

IFrom our first-hand experience, the handling of this species is extremely convenient. While we heavily promote the baculovirus expression system (BVES) for most of our custom protein production projects (we carried out one NIH project for producing human glycosylated cancer antigen proteins using a modified BVES recently), we now believe that there is a good chance that many of the proteins we have been producing could be produced in the protozoan system with potentially better efficiency.

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Wednesday, September 28th, 2011 Viruses and cells No Comments