Biopharmconsortium Blog

Expert commentary from Haberman Associates biotechnology and pharmaceutical consulting.

Monthly Archives: March 2012

“It’s not junk”–RaNA Therapeutics emerges from stealth mode with $20.7 million in venture funding-Part 2



This is Part 2 of the article on RaNA Therapeutics that we began on March 29, 2012.

Jeannie Lee’s research and RaNA’s technology platform

Jeannie Lee’s laboratory focuses on the study of the mechanism of X-chromosome inactivation in mammals. In X-chromosome inactivation, one of the two copies of the X chromosome present in the cells of female mammals is inactivated. The inactive X chromosome is silenced by packaging into transcriptionally inactive heterochromatin.  X-chromosome inactivation results in dosage compensation, the process by which cells of males and females have the same level of expression of X-chromosome genes, even though female cells have two X chromosomes and male cells have only one. In placental mammals such as mice and humans, the choice of which X chromosome will be inactivated is random, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants.

The Lee laboratory has focused on genes encoded by the X-chromosome whose actions coordinate X-chromosome inactivation. These genes  are contained in the 100 kilobase long X-inactivation center (Xic). One of these genes, Xist, encodes the lncRNA XIST, which as discussed in Part 1 of this article inactivates an X-chromosome by spreading along the X chromosome and recruiting the silencing factor PRC2. XIST is regulated in cis by TSIX, an antisense version of XIST which works to keep the active X-chromosome active. Tsix is in turn regulated by Xite (X-Inactivation intergenic transcription element), an upstream locus that harbors an enhancer that enables the persistence of TSIX expression on the active X chromosome. The mechanism by which Xite acts (including whether it acts via its RNA transcripts) is not clear. Xite and Tsix appear to regulate pairing between the two X chromosomes in a female cell, and determine which X chromosome will be chosen for inactivation. Several other recently discovered genes in the region of the Xic, which work via lncRNAs, also serve as regulators of XIST function. For example, the Rep A and Jpx genes, work via lncRNA transcripts to induce Xist. Thus Xist is controlled by positive and negative lncRNA-based switches–TSIX for the active X chromosome and JPX and REPA for the inactive X. Of these lncRNAs, REPA, XIST, and TSIX bind to and control PRC2.

In late 2010, the Lee laboratory published an article in Molecular Cell in which the researchers identified a genome-wide pool of over 9000 lncRNA transcripts that interact with PRC2 in mouse ES cells. Many of these transcripts have sequences that correspond to potentially medically-important loci, including dozens of imprinted loci (i.e., loci that are epigenetically modified such that only the paternal or maternal allele is expressed), hundreds of oncogene and tumor suppressor loci, and multiple genes that are important in development and show differential chromatin regulation in stem cells and in differentiated cells. The researchers obtained evidence that at least in one case, an RNAs works to recruit PRC2 to a disease-relevant genes, similar to PRC2 recruitment by XIST and HOTAIR. This case of specific PRC2 recruitment has not been previously known, suggesting that the researchers’ methodology could be used to discover new examples of PRC2 recruitment by lncRNAs.

Some of the PRC2-associated lncRNAs identified in the Molecular Cell report may be potential therapeutic targets and/or biomarkers. Overexpression of PCR2 proteins have been linked to various types of cancer, including metastatic prostate and breast cancer, and cancers  of the colon, breast, and liver. Pharmacological inhibition of PRC2-mediated gene repression was found to induce apoptosis in several cancer cell lines in vitro, but not in various types of normal cells. Induction of apoptosis in this system is dependent on reactivation of genes that had been repressed by PRC2. There is also evidence that PRC2-mediated gene repression may be linked to the maintenance of the stem-cell properties of cancer stem cells. These results suggest that at least in some cases, inhibition of PRC2-mediated gene repression–including via targeting lncRNAs that recruit PRC2 to critical genes–is a potential strategy for treating various types of cancer.

RaNA’s R&D strategy

Not much information is available about RaNA’s strategy.  However, according to the January 2012 Mass High Tech article, RaNA Therapeutics has licensed technology from Mass General Hospital based on Dr. Lee’s research. The company has also filed several patent applications, some of which are described as being very broad. This includes patent applications on the existence and method of use of thousands of lncRNA targets. However, Dr. Lee’s published patent applications currently include only three items involving the X-chromosome inactivation system or TERC. Presumably, the patent applications mentioned in the Mass High Tech article will be published at the end of the 18-month publication period for U.S. patent applications.

According to the Mass High Tech article, RaNA is in the process of narrowing down the diseases it will initially focus on. Likely areas will include genetic diseases, including diseases that result from haploinsufficiency. In haploinsufficiency, one allele of a gene is nonfunctional, so all of the protein coded by the gene is made from the other allele. However, this results in insufficient levels of the protein to produce a normal phenotype. RaNA intends to use its technology to increase expression of the functional gene, resulting in a adequate dosage of the protein for a normal phenotype.

RaNA intends to choose one indication out of a short list of 20 diseases for internal R&D, and to seek collaborations for other indications. Dr. Krieg says that he hopes to have a collaboration by the end of 2012, and also to have Investigational New Drug (IND)-enabling safety studies on its internal drug candidate by the end of the year as well.

As one might expect, RaNA will target the appropriate lncRNAs using oligonucleotides, similar to how RNAi companies target mRNAs. Dr. Krieg, an oligonucleotide therapeutic development veteran, recruited some of his old oligonucleotide team from Pfizer into RaNA, according to a Fierce Biotech article. Thus Dr. Krieg and his team can quickly get up and running in designing and testing oligonucleotide therapeutics, once RaNA selects the targets for its initial focus.

In the Mass High Tech article, Dr. Krieg says that he believes that “oligonucleotides are on the cusp of being recognized as the third leg of drug development,” along with small-molecule and protein therapeutics. However, as we discussed in our August 22, 2011 article on this blog, oligonucleotide drug development, as exemplified by RNAi and microRNA-based therapeutics, has run into several technological hurdles, especially those involving drug delivery. The August 2011 article cites an editorial by Dr. Krieg, in which he voices his optimism despite these hurdles.

Nevertheless, large pharmaceutical companies and investors have been moving away from the oligonucleotide field. This is exemplified by Alnylam’s January 20, 2012 restructuring, which cut one-third of its work force and focused the company on two of its Phase 1 programs. Having exhausted its ability to capture major Big Phama licensing and R&D deals, Alnylam has had to become a normal early-2012 biotech company and focus its strategy. (However, Alnylam did a $86.9 million public offering in February 2012.)

The emergence of RaNA, and its $20.7 million funding, thus swims against the tide of the general pessimism about oligonucleotide therapeutics of Big Pharmas, investors, and stock analysts. However, at least some oligonucleotide therapeutics will eventually emerge onto the market, and lncRNA regulation is likely to be crucial to many disease pathways. RaNA is thus the pioneering company in this field.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

“It’s not junk”–RaNA Therapeutics emerges from stealth mode with $20.7 million in venture funding-Part 1


XIST Source: Alexbateman


On January 18, 2012, start-up company RaNA Therapeutics (Cambridge, MA) emerged from stealth mode with 20.7 million in cash. The Series A venture funding was co-led by Atlas Venture, SR One, and Monsanto, with participation of Partners Innovation Fund.

RaNA will work on developing a technology platform that involves targeting long noncoding RNA (lncRNA), in order to selectively upregulate gene expression.

Arthur Krieg, M.D. will serve as RaNA’s President and CEO. He is the former Chief Scientific Officer of the now-closed Pfizer Oligonucleotide Therapeutics Unit, who later became an Entrepreneur in Residence at Atlas Venture (Cambridge, MA). Dr. Krieg was mentioned in two of our previous Biopharmconsortium Blog articles, dated February 15, 2011 and August 22, 2011.

Atlas quietly nurtured RaNA while working to complete the Series A venture round. According to a January 18, 2012 article in Mass High Tech, the company plans to move into about 9,000 square feet of space “somewhere in Cambridge” in early 2012.  RaNA has approximately a dozen employees.

According to the Mass High Tech article, RaNA’s platform is based on technology developed by scientific founder Dr. Jeannie Lee (Massachusetts General Hospital/Howard Hughes Medical Institute, Boston MA).  Drs. Lee and Krieg and Atlas Ventures are cofounders of RaNA.

This is Part 1 of our discussion of RaNA Therapeutics.

RaNA and “junk DNA”

RaNA’s focus is related to what has traditionally been called “junk DNA”. As shown by work on the Human Genome Project and other genomics studies, only about 2-3 percent of the human genome consists of protein-encoding genes. Genomics researchers had not been able to identify a function for most of the remaining 97-98% of the human genome. This gave rise to the idea that these sequences consisted of parasitic DNA sequences that had no function whatsoever. Most researchers thus called these sequences “junk DNA”. Some of the leading lights of the genomics field gave presentations in which they dismissed this DNA as “junk”, and they even proposed models for how this “junk DNA” might accumulate during evolution. Then they would go on to discuss the “interesting” 2-3 percent.

However, the “junk DNA” concept was not really established science, but a hypothesis. I–among a few others–would call these sequences “DNA of unknown function”.

In more recent years, many researchers showed that at least the vast majority of DNA of unknown function was transcribed. Then researchers found a function for a relatively small percentage of these sequences–they are precursors of microRNAs and other small regulatory RNAs. These RNAs are related to the phenomenon of RNA interference (RNAi), which has been the subject of much basic research, including the Nobel Prize-winning research of Drs. Andrew Fire and Craig Mello. RNAi is the basis for various therapeutic RNAi drug discovery and development efforts at such companies as Alnylam, Silence Therapeutics, Quark Phamaceuticals, Dicerna, and Santaris, as well as several large pharmaceutical companies.

The majority of DNA sequences of unknown function, however, are transcribed into lncRNA. As exemplified by the first article [“Quantity or quality?”, by Monika S. Kowalczyk and Douglas R. Higgs (University of Oxford)] in a point/counterpoint Forum published in the 16 February 2012 issue of Nature, many researchers postulate that at least most of these transcripts are nonfunctional. Transcription of these sequences might be, for example, at a low level, as the result of experimental artifacts or of exposure of sequences to the transcriptional machinery due to changes in chromatin during such processes as cell division or expression of nearby genes. This point of view moves the “junk DNA” hypothesis to the RNA level–now one might speak of “junk RNA”.

However, in the second article in the Nature Forum [“Patience is a virtue”, by Thomas R. Gingeras (Cold Spring Harbor Laboratory)], the author counsels “patience” in carefully unraveling the function, one by one, of each noncoding RNA (ncRNA) transcript. According to Dr. Gingeras’ article, there are currently some 161,000 human transcripts, 53% of which are ncRNAs. About 2% of these ncRNAs are precursors to microRNAs. Approximately 10% of the transcripts are lncRNAs that map to intergenic and intronic regions, and many of these transcripts have been implicated in regulation — both of locally and at a distance— of developmentally important genes. Another 16% of the ncRNAs are transcripts of pseudogenes — genes that appear to have lost their original functions during evolution. Some of the pseudogene transcripts have been shown to regulate gene expression by acting as decoys for microRNAs. Despite this progress in assigning functions to ncRNAs, no function has yet been found for the majority of these transcripts. However, these are early days in the ncRNA field, so patience and openness to new discoveries is advisable.

The same 16 February 2012 issue of Nature contains a “Nature Insight” supplement on “Regulatory RNA”. Of particular interest with respect to the functions of lncRNAs is the review by Mitchell Guttman (Broad Institute and MIT, Cambridge MA) and John Rinn (Broad Institute and Harvard, Cambridge MA), entitled “Modular regulatory principles of large non-coding RNAs”. Among the lncRNAs discussed in that review are the X-inactive specific transcript (XIST) (see the figure above) and the telomerase RNA component (TERC). Both of these lncRNAs were identified and their functions determined in the 1990s–XIST in 1991  and TERC in 1995. XIST is expressed exclusively from inactive X chromosomes and is required for X inactivation in mammals. TERC is an essential RNA component of telomerase, the enzyme that replicates chromosome ends (telomeres). At the same time as the functions of XIST and TERC were beginning to be unraveled, most researchers were continuing to dismiss ncDNA as “junk”. Should they have known better?

The Guttman and Rinn review discusses several other lncRNAs with known, important functions, all of which were discovered since the pioneering work on XIST and TERC. Among the genes that encode these lncRNAs are HOTAIR and HOTTIP, which affect expression of the HOXD and HOXA gene family, respectively. HOX genes are a superfamily of evolutionarily conserved genes that are involved the determination of the basic structure of an organism. They encode transcription factors that regulate target genes by binding to specific DNA sequences in enhancers. The large intergenic non-coding RNA-RoR (lincRNA-RoR) modulates reprogramming of human induced pluripotent stem cells (iPS cells, which were discussed in earlier articles on this blog). The lncRNA NRON regulates transcription factors of the NFAT (nuclear factor of activated T-cells) family, which are involved in regulating the immune response, as well as in the development of cardiac and skeletal muscle, and of the nervous system. These genes have also been implicated in breast cancer, especially in tumor cell invasion and metastasis.

A common theme in the function of several lncRNAs, as highlighted in the Guttman and Rinn review, is association of the lncRNA with a chromatin-regulatory protein complex. The lncRNA serves to guide the regulatory protein complex to specific regions of chromatin. The protein complex then modifies specific histones in the chromatin regions, resulting in silencing of target genes.

In particular, HOTAIR serves as a molecular scaffold that binds to two protein complexes. A 5′ domain of HOTAIR binds polycomb repressive complex 2 (PRC2), and a 3′ domain of HOTAIR binds the CoREST–LSD1 complex. This enables the targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation by PRC2 and histone H3 lysine 4 demethylation by LSD1. Both are required for proper repression of HOX genes.

XIST has at least two discrete domains, one involved in silencing (RepA) and the other in localization (RepC) of the XIST molecule on the X chromosome. The silencing domain RepA binds to PRC2, and the localization domain RepC binds to the YY1 protein and heterogeneous nuclear ribonucleoprotein U (hnRNP U).

The cases of HOTAIR and XIST are examples of how lncRNAs may function as molecular scaffolds of regulatory protein complexes. This may be general phenomenon, since a recent study by Drs. Guttman and Rinn and their colleagues indicates that about 30% of lincRNAs in mouse embryonic stem (ES) cells are associated with multiple regulatory complexes. In this study, the researchers found that RNAi knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of specific differentiation programs. Thus lincRNAs appear to have important roles in the circuitry controlling the pluripotent state of ES cells, and in commitment to differentiation into specific lineages.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

The Biopharmconsortium Blog hiatus to end soon!


As you all may have noticed, we have not had a post on the Biopharmconsortium Blog during the entire month of February, 2012.

This unintended hiatus happened because of the need to finish up other work.

We’re almost finished with these projects, and have several ideas for blog articles (including some articles that we had already started) in the hopper. So the Biopharmconsortium Blog hiatus will end soon.

Thank you very much for your patience, and for your continuing interest in our blog.