In Search of RNA Epigenetics: A Grand Challenge

  • Methylated riboA and riboC are the most commonly detected nucleobases in epigenetics research
  • Powerful new analytical methods are key tools for progress
  • Promising PacBio sequencing and novel “Pan Probes” reported   

In a Grand Challenge Commentary published in Nature Chemical Biology in 2010, Prof. Chuan He at the University of Chicago opined that “[p]ost-transcriptional RNA modifications can be dynamic and might have functions beyond fine-tuning the structure and function of RNA. Understanding these RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level.”

Like other scientists who get hooked by certain Grand Challenges, I became fascinated by this possibility of yet “new layers” of genetic regulation involving RNA, either as conventional messenger RNA (mRNA) or more recently recognized long noncoding RNA (lncRNA). Part of my intellectual stimulation was related to the fact that some of my past postings have dealt with both lncRNA as well as recent advances in DNA epigenetics, so the notion of RNA epigenetics seemed to tie these together.

After doing my homework on recent publications related to possible RNA epigenetics, it became apparent that this posting could be logically divided into commentary on the following three major questions: what are prevalent epigenetic RNA modifications, what might these do, and where is the field going? Future directions were addressed by interviews with two leading investigators: Prof. Chuan He, who is mentioned above, and Prof. Tao, who has been involved in cutting edge methods development.

RNA Epigenetic Modifications

More than 100 types of RNA modifications are found throughout virtually all forms of life. These are most prevalent in ribosomal RNA (rRNA) and transfer RNA (tRNA), and are associated with fine tuning the structure and function of rRNA and tRNA. Comments here will instead focus on mRNA and lncRNA in mammals, wherein the most abundant—and far less understood—modifications are N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

structures

Three Approaches to Sequencing m6A-Modified RNA

Discovered in cancer cells in the 1970s, m6A is the most abundant modification in eukaryotic mRNA and lncRNA. It is found at 3-5 sites on average in mammalian mRNA, and up to 15 sites in some viral RNA. In addition to this relatively low density, specific loci in a given mRNA were a mixture of unmodified- and methylated-A residues, thus making it very difficult to detect, locate, and quantify m6A patterns. Fortunately, that has changed dramatically with the advent of various high-throughput “deep sequencing” technologies, as well as other advances.

(1.) Antibody-based m6A-seq 

An impressive breakthrough publication in Nature in 2012 by a group of investigators in Israel reported novel methodology called m6A-seq for determining the positions of m6A at a transcriptome-wide level. This approach, which is a variant of methylated DNA immunoprecipitation (MeDIP or mDIP), combines the high specificity of an anti-m6A antibody with Illumina’s massively parallel sequencing of randomly fragmented transcripts following immunoprecipitation. These researchers summarize their salient findings as follows.

“We identify over 12,000 m6A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks—around stop codons and within long internal exons—and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m6A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signaling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m6A has a fundamental role in regulation of gene expression.”

Moreover, their concluding sentence refers back to He’s aforementioned Grand Challenge Commentary about RNA epigenetics in 2010, just two years earlier.

“The m6A methylome opens new avenues for correlating the methylation layer with other processing levels. In many ways, this approach is a forerunner, providing a reference and paving the way for the uncovering of other RNA modifications, which together constitute a new realm of biological regulation, recently termed RNA epigenetics.”

(2.) Promising PacBio Single-Molecule Real-Time (SMRT) Sequencing of m6A

In a previous post, I praised PacBio (Pacific Biosciences) for persevering in development of its SMRT sequencing technology that uniquely enables, among other things, direct sequencing of various types of modified DNA bases via differentiating the kinetics of incorporating labeled nucleotides. Attempts to extend the SMRT approach to sequencing m6A have been recently reported by PacBio in collaboration with Prof. Pan (see below) and others in J. Nanobiotechnology in April 2013. Using model synthetic RNA templates and HIV reverse transcriptase (HIV-RT) they demonstrated adequate discrimination of m6A from A, however, “real’ RNA samples having complex ensembles of tertiary structures proved to be problematic. Alternative engineered RTs that are more processive and accommodative of labeled nucleotides were said to be under investigation in order to provide longer read lengths and appropriate incorporation kinetics.

The authors are optimistic in being able to solve these technical problems, and concluded their report by stating:

  “[w]e anticipate that the application of our method may enable the identification of the location of many modified bases in mRNA and provide detailed information about the nature and the dynamic RNA refolding in retroviral/retro-transposon reverse transcription and in 3’-5’ exosome degradation of mRNA.”

Let’s hope that this is achieved soon!

(3.) Nanopore Sequencing of m6A?

It’s too early to be sure, but continued incremental advances in possible approaches to nanopore sequencing suggest applicability to m6A. As pictured below, Bayley and coworkers describe a method that uses ionic current measurement to resolve ribonucleoside monophosphates or diphosphates (rNDPs) in α-hemolysin protein nanopores containing amino-cyclodextrin adapters.

Taken from Bayley and coworkers in Nano Lett. (2013)

Taken from Bayley and coworkers in Nano Lett. (2013)

The accuracy of base identification is further investigated through the use of a guanidino-modified adapter. On the basis of these findings, an exosequencing approach for single-stranded RNA (ssRNA) is envisioned in which a processive exoribonuclease (polynucleotide phosphorylase, PNPase) presents sequentially cleaved rNDPs to a nanopore. Although extension of this concept to include m6A has yet to be demonstrated, earlier feasibility studies by Ayub & Bayley have shown discrimination of m6A (and other modified bases) from unmodified ribobases.

Two Probe-Based Methods for Detecting Specific m6A Sites

1.) “Pan Probes”

As the saying goes, “what goes around comes around”, and in this instance its repurposing 2’-O-methyl (2’OMe) modified RNA/DNA/RNA oligos. This general class of chemically synthesized chimeric “gapmers” was originally used for RNase H-mediated cleavage of mRNA in antisense studies. Very recently, however, Pan and coworkers have cleverly adapted these probes—which I like to alliteratively refer to as “Pan Probes”—to m6A detection in mRNA and lncRNA.

For details see SCARLET workflow; taken from Pan and coworkers RNA (2013)

Pan Probes are comprised of “7-4-7 gapmers” having seven 2’OMe RNA nucleotides flanking four DNA nucleotides, the latter of which straddle known (or suspected) m6A sites, as depicted in the cartoon shown. The indicated series of steps, which involve site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography, is thankfully called SCARLET by these investigators.

SCARLET was used by Pan and coworkers to determine the m6A status at several sites in two human lncRNAs and three human mRNAs, and found that the m6A fraction varied between 6% and 80% among these sites. However, they also found that many m6A candidate sites in these RNAs were not modified. Obviously, while much more work needs to be done to collect data for deciphering dynamic patterns and implications of m6A RNA epigenetic modifications, these investigators note that SCARLET is, in principle, applicable to m5C, pseudouridine, and other types of epigenetic RNA modifications.

Readers interested in designing and investigating their own Pan Probes can obtain these 7-4-7 gapmers by using TriLink’s OligoBuilder® and simply selecting “PO 2’OMe RNA” from the Primary Backbone dropdown menu, typing the first 7 bases in the Sequence box, selecting the 4 DNA bases from the Chimeric Bases menu and then typing the remaining 7 2’OMe RNA bases.

(2.) Probes for High-Resolution Melting

In a new approach very recently reported by Golovina et al. at Lomonosov Moscow State University, the presence of m6A in a specific position of mRNA or lncRNA molecule is detected using a variant of high-resolution melting (HRM) analysis applicable to, for example, single-nucleotide genotyping. The authors suggest that this method lends itself to screening many samples in a high-throughput assay following initial identification of loci by sequencing (see above). The method uses two labeled probes—one with 5’-FAM and another with 3’-BHQ1 (both available from Trilink’s OligoBuilder®)—that hybridize to a particular query position in a total RNA sample, as shown below for a 23S rRNA model system. The presence of m6A lowers the melting temperature (Tm), relative to A, with a magnitude that is sequence-context dependent.

Taken from Golovina et al. Nucleic Acids Res. (2013).

Taken from Golovina et al. Nucleic Acids Res. (2013).

The authors studied various probe-target constructs, and recommend 12–13-nt-long probes containing a quencher, and >20-nt long probes containing a fluorophore.  They also could advise that the quencher-containing oligonucleotide hybridizes to RNA such that m6A be directly opposite the 3′-terminal nucleotide carrying the quencher. The authors point out that relatively low-abundant, non-ribosomal targets need partial enrichment by, for example, simple molecular weight-based purification or commercially available kits. In this regard, they estimate that, if a particular type of mRNA was present at 10,000 copies per mammalian cell, 107 cells would be required to analyze m6A by this HRM method.

m5C Analysis by Sequencing of Bisulfite-Converted RNA

Selective reaction of bisulfite with C but not m5C in RNA, analogous to that long used for DNA, provides the basis for determining C-methylation status by sequencing. As detailed by Squires et al. in Nucleic Acids Res. in 2013, bisulfite-converted RNA can be sequenced by either of two methods: conversion to cDNA, cloning, and conventional sequencing, or conversion to a next-generation sequencing library. These authors described their salient findings as follows.

“We confirmed 21 of the 28 previously known m5C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions… Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.”

“Writing, Reading, and Erasing” RNA Epigenetic Modifications

Enzyme-mediated post-transcriptional RNA methylation (aka “writing”) and demethylation (aka “erasing”) are critical processes to identify and fully characterize in order to elucidate RNA epigenetics, and are formally analogous to those operative for DNA epigenetics.

RNA epigenetic “writing” mechanisms have focused on N6-adenosine-methyltransferase 70 kDa subunit, an enzyme that in humans is encoded by the METTL3 gene, and is involved in the posttranscriptional methylation of internal adenosine residues in eukaryotic mRNAs to form m6A. According to Squires et al., two m5C methyltransferases in humans, NSUN2 and TRDMT1, are known to modify specific tRNAs and have roles in the control of cell growth and differentiation.

As for “erasing”, in 2011, He’s lab discovered the first RNA demethylase, abbreviated FTO, for fat mass and obesity-associated protein, which has efficient oxidative demethylation activity targeting m6A in RNA in vitro. They also showed for the first time that this erasure of m6A could significantly affect gene expression regulation. In 2013, He’s lab discovered the second mammalian demethylase for m6A, ALKBH5, which affects mRNA export and RNA metabolism, as well as the assembly of mRNA processing factors, suggesting that reversible m6A modification has fundamental and broad functions in mammalian cells.

So, if Mother Nature evolved these mechanisms for writing and erasing RNA epigenetic modifications, what about the equally important, in between process of “reading” them? He and Pan and collaborators have very recently reported insights to such reading. They showed that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) “reader” protein to regulate mRNA degradation. They identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but also include non-coding RNAs, with a conserved core motif of G(m6A)C. They further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites. The carboxy-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2–mRNA complex to cellular RNA decay sites. These findings, they say, indicate that the dynamic m6A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

Expert Opinions of the Future for RNA Epigenetics

As I’ve said here before, there is no crystal ball for accurately predicting the future in science, although scientists do enjoy imagining that there is. Opinions of two “hands on” experts in the emerging field of RNA epigenetics are certainly of interest in this regard. Below are some comments offered by the aforementioned Prof. Tao Pan and Prof. Chuan He provided via an email interview in which I posed the question, ‘What do you see as the most important developments for RNA epigenetics?’ These experts have  thrown down the gauntlet, so to speak, by asserting RNA epigenetics as a Grand Challenge.

Prof. Tao Pan

Prof. Tao Pan

“In my opinion, the biggest current challenge for the field is to develop methods that can perturb m6A modification at specific sites to assess m6A function directly in specific genes. RNA interference or overexpression of an mRNA may simply decrease or increase modified and unmodified RNA alike. In a few cases, mutation of a known m6A site in an mRNA resulted in additional modification at a nearby consensus site, so that one cannot simply assume that mutation of a known site would not lead to cryptic sites nearby that may perform the same function. Further, functional understanding of a specific site should also take into account that all currently known m6A sites in mRNA and viral RNA are incompletely modified, so that one may need to explain why cells simultaneously maintain two RNA species that differ only at the site of m6A modification.”   

Prof. Chuan He

Prof. Chuan He

The m6A modification is much more abundant than other RNA modifications in mammalian and plant nuclear RNA and is currently the only known reversible RNA modification. The m6A maps of various organisms/cell types need to be obtained. High-resolution methods to obtain transcriptome-wide, base-resolution maps are important. A future focus should be to connect the reversible m6A methylation with functions, in particular, the studies of the reader proteins that specifically recognize m6A and exert biological regulation. The first example of the YTHDF2 work just published in Nature (above) is a good example. We believe many other reader proteins exist and impact almost all aspects of mRNA metabolisms or functions of lncRNA. 

Besides m6A, there are m5C, pseudoU, 2′-OMe, and potentially other modifications in mRNA and various non-coding RNAs (such as the recently discovered hm6A and f6A). The methods to map these modifications (except m5C) need to be developed and their biological functions need to be elucidated. 

Lastly, potential reversal of rRNA and tRNA modifications needs to be studied. As I stated in the Commentary in 2010, dynamic RNA modifications could impact gene expression regulation resembling well-known dynamic DNA and histone modifications. I think now we have enough convincing data to indicate this is indeed the case. The future is bright.”

Very bright, indeed! Your comments about this posting are welcomed.

Modified mRNA Mania

  • Biosynthetic modified mRNA for gene-based therapy without the gene!
  • AstraZeneca bets up to $420M on Moderna’s “messenger RNA therapeutics”
  • “Me-too” Pharma frenzy to follow?

In a perspective on gene therapy published in Science this year, Inder M. Verma starts by observing that the concept of gene therapy is disarmingly simple. Introduce a healthy gene in a patient and its protein product should alleviate the defect caused by a faulty gene or slow the progression of the disease. He then asks the rhetorical question: ‘why then, over the past three decades, have there been so few clinical successes in treating patients with this approach?’ The answer in part has to do with challenges for cell or tissue-specific delivery, which admittedly is an issue for virtually any type of therapeutic agent. There is also concern for adverse events generally ascribed to unintended vector integration leading to neoplasias. Nevertheless, according to Verma, the present clinical trials pipeline is jammed with more than 1700 (!) clinical trials worldwide, drawing on a wide array of gene therapy approaches for both acquired and inherited diseases.

In view of this scientifically laudable but undeniable—if not frustratingly—slow progress, it’s not surprising that various groups of investigators—and investors—have recently opted to pursue a strategy that eliminates a DNA-encoded gene entirely! Instead, biosynthetic mRNA is delivered in order to directly produce the desired therapeutic protein product—this is now being referred to as “mRNA therapeutics”.

Having said this, let’s consider some pivotal scientific publications, patents, and the emerging commercial landscape for what looks to be a very hot area for research and corporate competition.

Modified mRNA Therapeutic Vaccines

An excellent review published in 2010 by Bringmann et al. entitled RNA Vaccines in Cancer Treatment covers various approaches to using mRNA encoding for tumor-associated antigens to induce specific cytotoxic T lymphocyte and antibody responses. RNA-transfected dendritic cell vaccines have been extensively investigated and are currently in numerous clinical trials (the details for which can be found at the NIH ClinicalTrials.gov website by simply searching RNA vaccines).

Interestingly, clinical feasibility and safety assessment for direct intradermal injection of “naked” unmodified mRNA was reported back in 2008 by Weide et al., who removed metastatic tissue from each of 15 melanoma patients for total RNA extraction, reverse-transcription to cDNA, amplification, cloning, and transcription to produce unlimited amounts of copy mRNA.

Stabilizing unmodified mRNA by packaging in liposomes or forming complexes with cationic polymers has been widely investigated, as well as introducing chemical modifications to mRNA to make it more resistant against degradation and more efficient for translation. The latter includes elongation of the poly-A tail at the 3′-end of the molecule and modifications to the cap structure at the 5′-end. For example, if the original 7-methylguanosine triphosphate is replaced by an Antireverse Cap Analog (ARCA), the efficiency of transcription is strongly enhanced. To provide the immune system with even more potent signals, Scheel et al. modified mRNA with a phosphorothioate backbone in early commercial vaccine development work at CureVac GmbH (Tübingen, Germany) that continues today (see image below).

Effects of mRNA vaccines  (taken from an article in Drug Discovery & Development by Ingmar Hoerr, PhD, CEO and Cofounder of CureVac).

Effects of mRNA vaccines (taken from an article in Drug Discovery & Development by Ingmar Hoerr, PhD, CEO and Cofounder of CureVac).

In summary, in a 2013 review entitled RNA: The new revolution in nucleic acid vaccines, Geall et al. from Novartis Vaccines & Diagnostics (Cambridge, MA, USA) stated that “prospects for success are bright.” They site several reasons for this optimistic outlook including the potential of RNA vaccines to address safety and effectiveness issues sometimes associated with vaccines that are based on live attenuated viruses and recombinant viral vectors. In addition, methods to manufacture RNA vaccines are suitable as generic platforms and for rapid response, both of which will be very important for addressing newly emerging pathogens in a timely fashion. Plasmid DNA is the more widely studied form of nucleic acid vaccine and proof of principle in humans has been demonstrated, although no licensed human products have yet emerged. The RNA vaccine approach, based on mRNA, is gaining increased attention and several vaccines are under investigation for infectious diseases, cancer and allergy.

Modified mRNA for Expressing Clinically Beneficial Proteins

Dr. Katalin Karikó, Adjunct Associate Professor of Neurosurgery and Senior Research Investigator, Department of Neurosurgery, University of Pennsylvania (taken from upenn.edu).

Dr. Katalin Karikó, Adjunct Associate Professor of Neurosurgery and Senior Research Investigator, Department of Neurosurgery, University of Pennsylvania (taken from upenn.edu).

In a landmark publication by Karikó et al. in 2008, it was reasoned that the suitability of mRNA as a direct source of therapeutic proteins in vivo required muting its immunogenicity and boosting its effectiveness. Clues as to how this might be achieved were provided in their earlier work demonstrating the use of base-modified triphosphates to enzymatically synthesize in vitro mRNA having modified nucleosides [such as, pseudouridine (Ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine (s2U)] had greatly diminished immunostimulatory properties. They reasoned that, “if any of the in vitro transcripts containing nucleoside modifications would remain translatable and also avoid immune activation in vivo, such an mRNA could be developed into a new therapeutic tool for both gene replacement and vaccination”.

Using the aforementioned and other base-modified nucleotide triphosphates—all obtained from TriLink BioTechnologiesKarikó et al. found, surprisingly, that mRNA containing pseudouridine had a higher translational capacity than unmodified mRNA when tested in mammalian cells and lysates or administered intravenously into mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, and at each time-point there was more of the reporter protein when pseudouridine-containing mRNA was administered. Moreover, even at higher doses, only the unmodified mRNA was immunogenic. [Note: a fascinating follow-on publication provides a non-obvious—at least to me—molecular-level rationale for the surprising enhanced translation of pseudouridine-modified mRNA]. 

uridine

Uridine and pseudouridine differ in bonding to ribose but hydrogen-bond similarly to adenine. Pseudouridine is the most prevalent of the 100+ naturally occurring modified nucleosides found in RNA.

They concluded that, “[t]hese collective findings are important steps in developing the therapeutic potential of mRNA, such as using modified mRNA as an alternative to conventional vaccination and as a means for expressing clinically beneficial proteins in vivo safely and effectively.” Prior to publishing this pivotal report, Katalin Karikó and co-author Drew Weissman filed a patent application in 2006 entitled RNA containing modified nucleosides and methods of use thereof that was issued on October 2, 2012 as US 8,278,036 and is assigned to the University of Pennsylvania.

 

Blood Boosting with Erythropoietin

EPO stimulates the production of red blood cells (taken from proactiveinvestors.com via Bing Images)

EPO stimulates the production of red blood cells (taken from proactiveinvestors.com via Bing Images)

In a very persuasive demonstration of the real possibility of mRNA therapeutics, Karikó et al, reported in 2012 that non-immunogenic pseudouridine-modified mRNA encoding erythropoietin (EPO) was translated in mice and non-human primates. Indeed, a single injection of 100 ng (0.005 mg/kg) of HPLC-purified mRNA complexed to a delivery agent elevated serum EPO levels significantly and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma.

Rhesus macaque (taken from flickr.com via Bing Images)

Rhesus macaque (taken from flickr.com via Bing Images)

Using rhesus macaques (aka rhesus monkeys) they could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. Other researchers (Kormann et al.) independently used a single injection of modified murine mRNA to produce EPO in mice.

Kick-Start Cardiac Repair with VEGF-A

That’s the catchy title of a News & Views article in the October 2013 issue of Nature Biotechnology with an equally catchy byline that reads “[t]he survival of mice after experimental heart attack is greatly improved by a pulse of RNA therapy.” The featured report by Zangi et al., which is characterized as “a masterpiece of multidisciplinary studies…that will advance our thinking about therapeutic options in the cardiovascular arena,” is indeed impressive. These investigators report that intra-myocardial injections of vascular endothelial growth factor-A (VEGF-A) mRNA modified with 5-methylcytidine, pseudouridine, and 5’ cap structure resulted in expansion and directed differentiation of endogenous heart progenitors in a mouse model of myocardial infarction. They found markedly improved heart function and enhanced long-term survival of recipients. Moreover, “pulse-like” delivery of VEGF-A using modified mRNA was found to be superior to use of DNA vectors in vivo.

A heart attack (myocardial infarction) occurs when one of the heart's coronary arteries is blocked suddenly, usually by a blood clot (thrombus), which typically forms inside a coronary artery that already has been narrowed by atherosclerosis, a condition in which fatty deposits (plaques) build up along the inside walls of blood vessels (taken from drugs.com via Bing Images).

A heart attack (myocardial infarction) occurs when one of the heart’s coronary arteries is blocked suddenly, usually by a blood clot (thrombus), which typically forms inside a coronary artery that already has been narrowed by atherosclerosis, a condition in which fatty deposits (plaques) build up along the inside walls of blood vessels (taken from drugs.com via Bing Images).

Notwithstanding these promising results, the aforementioned News & Views article points out that microgram-scale doses of modified mRNA in mice used by Zangi et al. “would probably correspond to several hundred milligrams…in humans delivered in volumes that might exceed 10 ml per heart. In clinical practice, it would be very difficult to administer such volumes to infarcted hearts.” In my humble opinion, these are legitimate but purely hypothetical issues at this time and, given that it’s very “early days” for therapeutic modified mRNA technologies, it’s not unreasonable to assume that new modifications and/or improved delivery strategies can be developed to enable clinical utility.

From a technical perspective, this work by Zangi et al. involves a form of cell-free reprogramming and, as such, is a good segue into the next section. 

Modified mRNA for Cellular Reprogramming

In 2005, when I first heard of the concept of cellular reprogramming and dedifferentiation—which is to somehow coax a mature, differentiated cell to ‘run in reverse and go backwards biologically’ to a more primitive cell—my immediate impression as a chemist was this was impossible. Surely, I thought, this must violate the Second Law of Thermodynamics or, if not, is completely counterintuitive to how life works. Wow, was I wrong!

Reprogramming of differentiated cells to pluripotency is now firmly established and holds great promise as a tool for studying normal development.  It also offers hope that patient-specific induced pluripotent stem cells (iPSCs) could be used to model disease or to generate clinically useful cell types for autologous therapies aimed at repairing deficits arising from injury, illness, and aging. Induction of pluripotency was originally reported by Takahashi & Yamanaka by enforced retroviral expression of four transcription factors, KLF4, c-MYC, OCT4, and SOX2 (aka “Yamanaka factors”)—collectively abbreviated as KMOS. (TriLink sells these and other factors used to direct cell fate.) Viral integration into the genome initially presented a formidable obstacle to therapeutic use of iPSCs. The search for ways to induce pluripotency without incurring genetic change has thus become the focus of intense research effort.

Consequently, much attention has been given to the 2010 publication by Warren et al. entitled Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. In this work complete substitution of either 5-methylcytidine for cytidine or pseudouridine for uridine in protein-encoding transcripts markedly improved protein expression, although the most significant improvement was seen when both modifications were used together. Transfection of modified mRNAs encoding the above mentioned Yamanaka factors led to robust expression and correct localization to the nucleus. Expression kinetics showed maximal protein expression 12 to 18 hours after transfection, followed by rapid turnover of these transcription factors. From this it was concluded that daily transfections would be required to maintain high levels of expression of the Yamanaka factors during long-term, multifactor reprogramming regimens.

They went on to demonstrate that repeated administration of modified mRNA encoding these (and other) factors led to reprogramming various types of differentiated human cells to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. Importantly, this simple, non-mutagenic, and highly controllable technology was shown to be applicable to a range of tissue-engineering tasks, exemplified by mRNA-mediated directed differentiation of mRNA-generated iPSCs to terminally differentiated myogenic (e.g. heart muscle) cells.

Modified mRNA reprogramming fibroblasts into induced pluripotent cells for directed differentiation into myofibers, according to Warren et al. in Cell Stem Cell (2010)

Modified mRNA reprogramming fibroblasts into induced pluripotent cells for directed differentiation into myofibers, according to Warren et al. in Cell Stem Cell (2010)

Warren et al. concluded that “we believe that our approach has the potential to become a major enabling technology for cell-based therapies and regenerative medicine.”  According to the Acknowledgements section of this 2010 publication, corresponding author Derrick J. Rossi recently founded a company, ModeRNA [sic] Therapeutics, dedicated to the clinical translation of this technology.” That, we shall see below, has had stunning commercial investment consequences.

By the way, and not surprisingly, Rossi & Warren filed a U.S. patent application in 2012 claiming, among other things, iPSCs induction kits using 5-methylcytidine- and pseudouridine-modified mRNA encoding KMOS human cellular reprogramming factors.

AstraZeneca’s Big Bet on Moderna’s Modified-mRNA Therapeutics

AstraZeneca aims to use Moderna Therapeutics’ modified-messenger RNA technology to develop and commercialize new drugs for cancer and serious cardiovascular, metabolic, and renal diseases, under a multi-year deal that could net Moderna more than $420 million. Moderna is also eligible for royalties on drug sales ranging from high single digits to low double digits per product.

AstraZeneca—ranked 7th in sales in 2010 among the world’s pharmaceutical companies—has the option to select up to 40 drug products for clinical development of what the companies are calling messenger RNA Therapeutics™, which could dramatically reduce the time and expense associated with creating therapeutic proteins using current recombinant technologies, the companies say. Moreover, “where current drug discovery technologies can target only a fraction of the disease-relevant proteins in the human genome, we have the potential to create completely new medicines to treat patients with serious cardiometabolic diseases and cancer,” AstraZeneca CEO Pascal Soriot said in a statement. Mr. Soriot, who had been a senior executive at Roche, very recently joined AstraZeneca, which said it would reorganize R&D and eliminate 1,600 jobs by 2016 as part of a plan to address issues related to failures in clinical trials of several drugs just as big sellers like the antipsychotic Seroquel and the heartburn drug Nexium have lost or are about to lose patent protection.

Moderna, based in Cambridge, Massachusetts, is privately held and was founded in 2010 by Flagship VentureLabs in association with leading scientists from Boston Children’s Hospital and Massachusetts Institute of Technology. Moderna has developed a broad intellectual property estate including 144 patent applications with 6,910 claims ranging from novel nucleotide chemistries to specific drug compositions, according to its website.

DARPA also Bets Big on Moderna’s Modified-mRNA Therapeutics

As the saying goes, “when it rains it pours”, and for Moderna it’s pouring money!

On October 2nd, Moderna announced that the U.S. Defense Advanced Research Projects Agency (DARPA)—whose most successful bets so far have been internet technologies—has awarded the company up to $25 million for R&D using its modified-mRNA therapeutics platform as a “rapid and reliable way to make antibody-producing drugs to protect against a wide range of now and emerging infectious diseases and engineered biological threats.” The statement goes on to say that Moderna’s approach can “tap directly into the body’s natural processes to produce antibodies without exposing people to a weakened or inactivated virus or pathogen, as in the case with the vaccine approaches currently being tested.”

The grant could support research for up to 5 years to advance promising antibody-producing drug candidates into preclinical testing and human clinical trial. The company also received a $700,000 ‘seeding’ grant from DARPA in March to begin work on the project.

If you’re interested in some of the possible ideas associated with the project, go to the 2013 patent application by Moderna entitled Methods of responding to a biothreat, which even envisages a portable, battery operated device for synthesizing modified mRNA. Oh well, never let it be said that DARPA fears a risky bet; on the other hand, since DARPA’s “playing with house money” (aka our taxes!), I suppose it’s easy for them. Let’s hope they/we all win.

Other Commercial Players

In addition to TriLink’s mRNA products, related services, and new cGMP facility, there are other companies to mention here, which I’ll do in alphabetical order.

  • Acuitas Therapeutics has compared the effectiveness of its lipid nanoparticle (LNP) carriers in vivo with the most potent delivery systems reported in the scientific literature, and found that Acuitas LNPs demonstrate much greater luciferase expression in the liver after systemic administration.
  • CureVac is combining both the antigenic and adjuvant properties of mRNA to develop novel and effective mRNA vaccines. CureVac is currently developing therapeutic mRNA vaccines in oncology and therapeutic/prophylactic vaccines for infectious diseases. Information on five of its clinical studies is available at ClinicalTrials.gov.
  • Dendreon has a U.S. patent application for a method to make dendritic cell vaccines from embryonic stem cells that are genetically modified with mRNA encoding tumor antigen. However, no mRNA-searchable items are currently listed on Dendreon’s website.
  • In-Cell-Art is investigating new and improved nanocarriers for mRNA vaccines, and has collaborated with Sanofi Pasteur and CureVac in DARPA-funded studies.
  • Mirus Bio offers a TransIT®-mRNA Transfection Kit for high efficiency, low toxicity, mRNA transfection of mammalian cells, as described by Karikó et al.

Also noteworthy, the 1st International mRNA Health Conference recently held on October 23-24 at the University of Tübingen included talks by numerous key scientists in academia and industry that are well worth looking at in the Conference Program.

In conclusion, I hope that you found this emerging area of modified mRNA therapeutics as interesting and exciting as I did in researching this blog posting, and I welcome your comments.

Postscript

After finishing the above blog, I came across these additional publications on possible mRNA therapies.

Huang and coworkers reported earlier this year that systemic delivery of liposome-protamine-formulated modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy was significantly more effective than plasmid DNA in a therapeutic model of human lung carcinoma in xenograft-bearing nude mice.

Zimmermann et al. reported successful use of mRNA-nucleofection for overexpression of interleukin-10 in murine monocytes/macrophages for anti-inflammatory therapy in a murine model of autoimmune myocarditis. [Note: for a related report on mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation see Levy et al.]

Cystic Fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Bangel-Ruland et al. reported in vitro results indicating that CFTR-mRNA delivery provided a novel alternative for cystic fibrosis “gene therapy”.

The Buzz on the Cut: From Dream to Reality

Targeted Genome Engineering with Zinc-finger Nucleases, TALENs and CRISPR

Targeted genome editing tools such as meganucleases, zinc-finger nucleases, TALENs and CRISPR are among the hottest topics in cell and gene therapy. Dr. Anton McCaffrey, Principal Scientist at TriLink and expert in these areas, gives herein his overview after attending the recent American Society for Gene and Cell Therapy Meeting (May 2013) and the International Society for Stem Cell Research Meeting (June 2013) where there were a number of exciting talks discussing applications of this technology.

amccaffrey


Dr. McCaffrey received his PhD in Biochemistry from the University of Colorado at Boulder in 1999. During his postdoctoral fellowship at Stanford he developed gene therapeutics for hepatitis B and C. He was then Assistant Professor at
University of Iowa where he focused on the role of microRNAs during the pathogenesis of hepatitis C virus and developed RNA interference and zinc-finger nuclease based therapeutics for treatment of hepatitis B virus. Now he manages the RNA Transcription product line at TriLink.

So what is targeted genome engineering with nucleases and why would you want to do this?  The primary goal in cells or animals is to create a specific, localized double-stranded DNA break and then to: 1. correct the sequence of a defective targeted gene, 2. knock in a specific gene mutation to create a disease model or 3. knock out a gene. The basic idea is to rationally design artificial restriction enzymes that recognize a specific location within the DNA genome of a cell or organism and catalyze a double stranded break at this location (Figure 1).

In the first two cases, where the target gene is to be specifically edited, the nuclease(s) are co-transfected with an exogenous donor DNA molecule. This donor DNA contains arms, which share homology with the target loci and will direct homologous recombination at the targeted cut site. The sequence of the donor DNA replaces that of the endogenous locus at one or both alleles. So, for example, a wild-type donor sequence can be used to replace a mutated gene sequence to correct a genetic disease.

If one wishes to inactivate a gene using these technologies, an exogenous donor template is not included. In the absence of a donor, the cell uses non-homologous end joining to repair the double stranded break. At a high frequency, this process introduces deletions and insertions in the gene, which changes the reading frame and inactivates the gene.

Until the advent of these technologies, it was impossible to make transgenic animals other than mice. Using targeting genome engineering is now possible to make transgenic rats, pigs, ferrets and plants. As will be discussed below, advances in messenger RNA (mRNA)-based gene therapy are converging with advances in targeted genome engineering to enable efficient, yet transient expression of designer nucleases without risk of undesired integration of the nuclease expression vector.

Figure 1.  Nuclease Mediated Double Stranded Breaks Stimulate Homologous Gene Replacement or Targeted Gene Inactivation.  If targeted nucleases are co-transfected with a homologous donor DNA fragment, homologous recombination replaces defective DNA with a corrected sequence (left).  In the absence of a DNA donor fragment, non-homologous end joining repairs the break, but with frequent insertions and deletions, thus inactivating the gene.


Figure 1. Nuclease Mediated Double Stranded Breaks Stimulate Homologous Gene Replacement or Targeted Gene Inactivation. If targeted nucleases are co-transfected with a homologous donor DNA fragment, homologous recombination replaces defective DNA with a corrected sequence (left). In the absence of a DNA donor fragment, non-homologous end joining repairs the break, but with frequent insertions and deletions, thus inactivating the gene.

Meganucleases

Techniques for making targeted nucleases are rapidly evolving.  Initial attempts to engineer designer restriction nucleases to target new sequences revolved around changing the specificity of naturally occurring nucleases such as meganucleases.  Meganucleases are restriction enzymes with long recognition sites (12-40 nucleotides). These nucleases could be engineered to recognize related sequences in genomes and cleave them.  However, only a small number of sites could be targeted using this approach (refs A-C).

Zinc-finger nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) were the next major advance in the field. Zinc fingers are the most common DNA binding motif in mammalian transcription factors. These sequence specific binding domains can be engineered to bind to novel DNA sequences. Zinc-fingers can be turned into nucleases by fusing them to non-specific cleavage domains, such as the FokI nuclease. FokI cleaves as a dimer, so pairs of ZFNs are designed to bind to adjacent sites in the genome to allow FokI dimer formation and double stranded DNA cleavage (Figure 2). A number of laboratories published design rules that serve as a starting point to engineer ZFNs with novel DNA binding specificities (refs N-R). In reality, actual binding specificity is context dependent. Several selection protocols in cells also exist for identifying novel ZFNs. ZFNs have been successfully used to modify the genomes of Drosophila, C. elegans, zebrafish and rats (refs D-M). However, identification of functional ZFNs remains challenging and most ZFNs have emerged from a small number of laboratories with specialist skills.

 Figure 2. Zinc-Finger Nucleases Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]


Figure 2. Zinc-Finger Nucleases Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]

Transcription activator-like effector nuclease (TALENs)

In the last few years, TALENs have emerged as a more generally accessible alternative to ZFNs. Like ZFNs, TALENs utilize a modular DNA binding motif (TALE) that can be modified to introduce new DNA binding specificities. TALENs consist of multiple repeat variable diresidues (RVDs) which each specify binding to a single nucleotide (Refs S-U).  TALEN arrays are made by stringing together RVDs in a specific order to provide specificity and binding affinity to novel DNA sequences. Commonly, engineered TALE sequences are fused to non-specific cleavage domains such as FokI. As with ZFNs, TALENs function as pairs bound to adjacent DNA sequences. Unlike ZFNs, TALENs are not as prone to sequence context effects, which greatly complicate the de novo design of ZFNs. This has made them much more accessible to the general scientific community. A number of groups have published TALEN assembly protocols that allow assembly of these repetitive sequences, including one popular open source assembly method is known as Golden Gate (Refs V-Y).

 Figure 3. TALENs Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]


Figure 3. TALENs Bind as Dimers to Cut Double Stranded DNA. Adapted from Gaj et al.Trends Biotechnol. 2013 May 8. [Epub ahead of print]

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

The newest kid on the genome-engineering block is CRISPR. CRISPR is a bacterial immune system in which bacteria sample the DNA of pathogens, integrate foreign DNA into their genome in specialized repeat structures, and then use these sequences to produce Guide RNAs that direct cutting of homologous pathogenic DNA sequences. To some degree this is reminiscent of RNA interference in mammals. Once the target site has been delineated by the RNA guide sequence, Cas proteins (CRISPR-associated proteins) do the cutting. A number of groups have adapted this system to create RNA directed genome engineering tools (refs Z-CC) (Figure 4). This new system has generated considerable interest since recognition of the target DNA sequence to be cut is RNA mediated rather than protein mediated. DNA cleavage is carried out by the expressed Cas9 protein. With ZFNs and TALENs, if you wish to target a new site, you have to identify and synthesize two new proteins. With CRISPR, you use the same Cas9 protein each time and just alter the sequence of the guide RNA. Stay tuned for head to head comparisons of the efficiency and specificity of ZFNs, TALENs and CRISPR that are about to be published.

 Figure 4. CRISPR is an RNA Guided Genome Engineering System.  Figure adapted from DiCarlo et al. Nucleic Acids Research, 2013, Vol. 41, No. 7.


Figure 4. CRISPR is an RNA Guided Genome Engineering System. Figure adapted from DiCarlo et al. Nucleic Acids Research, 2013, Vol. 41, No. 7.

Modified mRNA for Transient Expression in Genome Engineering

In each of the three systems described above, one needs to express one or two proteins inside cells or an organism.  Plasmids and viral vectors have been used to achieve this, but these carry a risk. Double stranded DNA breaks catalyze insertion of DNA at the cut site.  At some substantial frequency, the protein expression vectors can integrate at the cut site. These vectors necessarily carry eukaryotic promoters, which can lead to continuous expression of the nuclease or the expression of previously silent sequences. For clinical applications this can be a major issue. One also needs to consider off-target cleavage of by engineered nucleases. Since ZFNs have been around longer than TALENs or CRISPR, the most data exists for ZFNs. It is clear that ZFNs can cut at pseudo-sites that resemble the chosen target site.For this reason, transient expression of nucleases is desirable. Many in the ZFN and TALEN field have moved to expression of these nucleases from synthetic mRNAs because they are transient and have no risk of insertion.  Synthetic mRNAs, which mimic fully processed, capped and polyadenylated mRNAs, can be produced in large quantities by in vitro transcription. Transfected mRNAs made with adenine, cytosine, guanine and uracil are recognized as pathogens by innate immune sensors such as Toll-like receptors, RIG-I and PKR. Kariko et al. showed that mRNAs could be made much less immunogenic and non-toxic by substitution of cytosine and uridine with 5-methylcytosine and pseudouridine (ref DD). Custom syntheses of milligram to gram amounts of 5-methylcytosine and pseudouridine modified mRNAs can be ordered from TriLink BioTechnologies. Cas9 mRNA is also available as a catalog item.

Conclusions

In recent years, designer genome engineering has gone from dream to reality. New editing systems are taking this from the realm of a few elite laboratories and companies to democratizing it for the masses. Concurrent advances in mRNA gene therapy are providing safe and effective delivery systems for expressing the necessary components in cells and animals. There is now huge interest in using targeted genome engineering in patient derived somatic cells and stem cells. Rather than simply knocking genes in or knocking them out, we may now be able to actually correct monogenic genetic disorders.  Clinical trials are currently under way to determine if ZFNs can be used to inactivate the CCR5 HIV co-receptor to make patient T-cells immune to HIV. These technologies will also enable facile creation of disease models in species other than mice. The future is bright for targeted genome engineering. That’s the buzz on the cut.

A sincere thanks to Anton McCaffrey for providing this update on truly exciting trends in nucleic acid-based technologie. As always, I welcome comments and discussions.

References

A. I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Cohen-Tannoudji M, Robine S, Choulika A, et al. Mol Cell Biol 1998;18:1444-8.

B. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells. Choulika A, Perrin A, Dujon B, Nicolas JF. C R Acad Sci III 1994;317:1013-9.

C. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Choulika A, Perrin A, Dujon B, Nicolas JF. Mol Cell Biol 1995;15:1968-73.

ZFNs modifying different organisms

D. Efficient gene targeting in Drosophila with zinc-finger nucleases. Beumer K, Bhattacharyya G, Bibikova M, Trautman JK, Carroll D. Genetics 2006;172:2391-403.

E. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Beumer KJ, Trautman JK, Bozas A, et al. Proc Natl Acad Sci U S A 2008;105:19821-6.

F. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Bibikova M, Golic M, Golic KG, Carroll D. Genetics 2002;161:1169-75.

G. Genetic Analysis of Zinc-finger Nuclease-induced Gene Targeting in Drosophila. Bozas A, Beumer KJ, Trautman JK, Carroll D. Genetics 2009.

H. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Morton J, Davis MW, Jorgensen EM, Carroll D. Proc Natl Acad Sci U S A 2006;103:16370-5.

I. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Doyon Y, McCammon JM, Miller JC, et al. Nat Biotechnol 2008;26:702-8.

J. Zinc finger-based knockout punches for zebrafish genes. Ekker SC. Zebrafish 2008;5:121-3.

K. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). Foley JE, Yeh JR, Maeder ML, et al. PLoS ONE 2009;4:e4348.

L. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA. Nat Biotechnol 2008;26:695-701.

M. Knockout rats via embryo microinjection of zinc-finger nucleases. Geurts AM, Cost GJ, Freyvert Y, et al. Science 2009;325:433.

ZFN design rules

N. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Segal DJ, Dreier B, Beerli RR, Barbas CF, 3rd. Proc Natl Acad Sci U S A 1999;96:2758-63.

O. Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains. Dreier B, Segal DJ, Barbas CF, 3rd. J Mol Biol 2000;303:489-502.

P. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. Liu Q, Xia Z, Zhong X, Case CC. J Biol Chem 2002;277:3850-6.

Q. Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF, 3rd. J Biol Chem 2001;276:29466-78.

R. Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. Dreier B, Fuller RP, Segal DJ, et al. J Biol Chem 2005;280:35588-97.

TALENS

Breaking the code of DNA binding specificity of TAL-type III effectors. S. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A. and Bonas, U. Science 2009;326,1509-12.

T.  The crystal structure of TAL effector PthXo1 bound to its DNA target. Mak, A.N., Bradley, P., Cernadas, R.A., Bogdanove, A.J. and Stoddard, B.L. Science 2012;335, 716-9.

U. A simple cipher governs DNA recognition by TAL effectors. Moscou, M.J. and Bogdanove, A.J. Science 2009;326,1501.

Golden gate

V. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., Voytas, D.F., Geissler et al. Nucleic Acids Res 2011;39,e82.

W. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Li, T., Huang, S., Zhao, X., Wright, D.A., Carpenter, S., Spalding, M.H., Weeks, D.P. and Yang, B., Morbitzer et al. Nucleic Acids Res 2011;39, 6315-25.

X. A modular cloning system for standardized assembly of multigene constructs. Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. PLoS One 2011;6, e16765.

Y. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G.M. and Arlotta, P. Nat Biotechnol 2011;29,149-53.

CRISPR

Z. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Jinek, M; Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. Science 2012;PMID 22745249.

AA. Multiplex genome engineering using CRISPR/Cas systems. Cong, Le; Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science 2013;PMID 23287718.

BB. RNA-guided human genome engineering via Cas9. Mali, P; Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. Science 2013;PMID 23287722.

CC. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang, H; Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. Cell 2013;PMID 23643243.

Modified mRNA

DD. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D. Mol Ther. 2008;Nov;16(11):1833-40.