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

 

XIST Source: Alexbateman http://bit.ly/GZNTZg

 

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.

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