Sunday, October 12, 2014

Beyond Small Molecules--Gene Editing Strategies Provide Real Hope to Truly "Leave No Mutation Behind." NACFC 2014

Successful rescue of CFTR function with small molecules like Kalydeco, or corrector/potentiator combination therapies (VX-809/VX-770) hold a lot of promise for the immediate future of altering the course of CF disease.  The tricky part about small molecule therapy, is that different mutations produce their own unique issues.  As we know already, Kalydeco monotherapy is effective for a small percentage of CF mutations, but produces little or no effect at all, in others.  Finding novel combinations of correctors and potentiators to successfully rescue robust amount of CFTR protein function for the myriad of mutations will not be a simple task.  Each year, I hear about dozens of new corrector and potentiator compounds that may be combined in different ways to successfully affect CFTR rescue.  After seeing the potential of this type of therapy in my own son, I am anxious to see more small molecules treatments successfully reach the market.

The small molecule approach can be a very powerful way to impact CFTR function, but still represents a temporary fix for a permanent problem.  For patients like Brady, Kalydeco might be considered a "one day cure."  As long as he maintains sufficient levels of Ivacaftor pumping through his veins, his body doesn't really realize it has CF..., BUT, if he were to stop swallowing those expensive little blue darlings, his CF symptoms would return quickly.  Because of the temporary nature of the effects, small molecules don't really fit the bill as a true cure.  While the CFF has millions of dollars invested in the development of new small molecules, they are also looking even further toward the future. Ultimately, we would all love to see a LIFETIME cure, rather than a ONE DAY cure.  True to the "venture philanthropy" model that The CF Foundation has made famous, they are poised to make considerable investments in new exciting fields of research that could yield that lifetime cure that we all dream of, for each and every person with cystic fibrosis--regardless of their genetic mutations.  I was disappointed that the UK Gene Therapy Trial results were not presented at this meeting as scheduled, but there are plenty of other new technologies to get REALLY excited about.

Symposium Session 8: Gene Editing Strategies for Therapy and Research

In this session, two technologies were described to manipulate genes such as CFTR. What makes these techniques so attractive is that they have the potential to be tailored to treat diverse cystic fibrosis mutations. These techniques can be thought of as "mutation agnostic," and could provide functional results for the multitudes of CF mutations, no matter how rare or complex the dysfunction.

1) Genome editing by the CRISPR/Cas9 system
2) RNA editing--where mutations in messenger RNA are corrected without modification of the genomic DNA

Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of CF Patients
Jeffrey Beekman

To perform this study, adult intestinal stem cells were obtained from rectal biopsies of adults with CF.  Just to be perfectly clear--embryonic stem cells were not required in this research.  The tissue samples obtained from the rectal biopsies were then taken into the lab, where they were manipulated to create intestinal organoids for experimentation.  Most people have heard a lot about changes in sweat chloride levels as a measure of CFTR function.  Over the last several years, I've seen more and more evidence that, while correlated with CFTR function, sweat chloride levels may not paint the most accurate picture of how much functional CFTR is being rescued.  A better way to directly measure CFTR channel function is to examine intestinal organoid swelling.  

This slide details the methods used to grow organoids from adult stem cells. 

CFTR function can be directly measured via the amount of swelling that intestinal organoid cells exhibit.  The amount of swelling can be directly correlated to CFTR function.  This has been found to be a much more accurate measure of CFTR channel activity than sweat chloride measurements.

 For the record, CRISPR stands for clustered regularly interspaced short palindromic repeats--specific targets on a gene, containing short repetitions of base sequences, followed by short segments of "spacer" DNA. Cas9 is a single DNA targeting enzyme that serves to "code" for proteins related to CRISPRS.  In simpler terms, the CRISPR can be thought of as the specific portion of DNA targeted for alteration (the section where we find the presence of the CF mutation).  Cas9 can be thought of as a tiny pair of scissors, programmed to make a cut in the strand of DNA at precisely the right spot.

Here, the CRISPR-Cas9 gene editing system was used to repair the CFTR-F508 target.  Once the double strand cut is made in the DNA by Cas9, the DNA undergoes homologous recombination--or automatic repair to "fix" the break. After recombination, the repaired DNA no longer contains the problematic CF mutation.

Using this method, researchers were able to restore functional swelling of the organoids in homozygous DF508, indicating restored CFTR function.

This work is very preliminary...but extremely exciting.  This experiment serves as proof-of-concept for "regenerative medicine" approaches of CF using gene-corrected adult stem cells.  In addition, this study shows that intestinal organoid swelling can be an effective mode of measuring CFTR function for future studies.  At this point, researchers will need to work on improving the efficiency of correction, which is still very low.  They seem confident that refinement of technique can produce much higher efficiency in future studies.

Correction of Genetic Mutations By Site-Directed RNA Editing
Joshua Rosenthal

The description of RNA editing involves a lot of technical jargon, so I am going to lay this out in two totally different ways—first the scientific version, and then my layman’s translation. 

Version 1:
Adenosine deaminases that act on RNA (ADARS) are a family of enzymes whose activity resembles a natural form of targeted mutagenesis.  Biochemically, ADARS convert adensosine to inosine—a nucleotide that is read as guanosine during translation.  ADARS are modular enzymes with distinct domains that perform different functions.  At one terminus, they contain a deaminase domain that catalyzes the deamination of adenosine to inosine.  At the other end, a number of double-stranded RNA binding motifs (dsRBMs) are found.  These dsRBMs bind to the tertiary structures in pre-mRNAs.  It is problematic to manipulate the ADARS targeting mechanism, so researchers decided to replace ADAR’s dsRBMs (the binding end of the enzyme) with a more easily manipulated, antisense RNA oligonucleotide—which could be easily directed to bind with any primary sequence along an RNA.  Researchers found, that when this antisense RNA oligonucleotide was joined with the dsRBM (via a small bacteriophage binding protein), they could selectively target and edit a single adenosine.  When editing occurs in mRNAs, codons can be recoded and the changes can alter the protein function.  For example, mutations which cause premature termination codons (UAA, UGA, UAG) could be recoded to tryptophan (UGG) to achieve successful protein read-through.  To test their editing system, and provide proof of concept for encoding CFTR, researchers selected the W496X mutation, which contains an early “stop” codon.  In vitro, they showed that their system of directed editing could correct W496X with near perfect efficiency.  In Xenopus oocytes, the genetically encoded version of their editase corrected CFTR mRNA, restored full-length protein, and reestablished functional chloride currents across the plasma membrane.  In human cell lines (grown in lab), their editing system was able to correct a non-functional version of enhanced green fluorescent protein (eGFP) with a premature termination codon.  The next step is to try and correct endogenous CFTR W496X, and other CFTR mutations caused by G-to-A transition, in epithelial cell lines. 

Version 2:
Since I am sitting in the airport, I will also describe RNA editing like this—Imagine DNA as our flight map.  This “map” tells the pilot what the final destination is, and how to get there.  The RNA can be thought of as the “pilot” of our flight, whose job is to follow the map, and deliver passengers where they are supposed to go.  Of course, the pilot (RNA) knows all the details about the trip: how high to take the plane, when to make turns, and how to land at the final destination.  The problem is that sometimes the pilot shows up drunk, and causes the flight to crash and burn.  In that case (as with premature stop codon CFTR mutations), none of the passengers reach their destination.  ADARS (paired with the antisense RNA oligonucleotide) can be thought of as “hijackers,” secretly coming onboard and forcefully changing the details of the flight plan.  They mask the pilot, steal his uniform, pilot hat, and cute little wings (to look “official” and keep the passengers calm) and take the plane to a different destination than originally planned.  Fortunately, the hijackers (in the case of RNA editing) are actually pretty cool (especially compared with to the passed out drunk previously in charge), and instead of taking the blissfully unaware passengers to their originally scheduled work conference in Shittsville, (no CFTR protein produced) decide to redirect the flight to an all-inclusive resort in The Cayman Islands (functional, full-length CFTR). 
I hope I was able to convey the fundamental concept behind this new research, because site directed RNA editing is truly a very promising and exciting strategy to correct a broad variety of genetic mutations, in both CFTR as well as other proteins.  The correction of genetic mutations at the mRNA level is attractive for several reasons. 

1) Compared to DNA, mRNA is easily accessible (genomic DNA is sequestered in the nucleus and tightly bound by histones).

2)  Mature mRNA is in the cytoplasm.  The delivery of a site-directed editor to mRNA would only entail crossing a single membrane.

3) Because RNA is transient (where DNA is not), off-target edits (targeting mistakes) are less dangerous.

4) Site-directed RNA editing does not affect mRNA expression level (expression level must be precise—as both under-expression or over-expression can result in disease). 
I've never actually visited The Cayman Islands before...but it seems like a really nice place.


  1. "This experiment serves as proof-of-concept for "regenerative medicine" approaches of CF using gene-corrected adult stem cells."

    Just one detail. This is wrong. Correction therapy will not be done by administering stem cells. This has been tried and has shown poor results. Correction therapy will be done by administering the correctional (?) agents and delivering them to the cells in the body.
    This is also not regenerative therapy. Correcting the cells will not undo the damage that is already done. That part will require other solutions.

    1. The quoted statement came directly from the author, and can be found in Pediatric Pulmonolgy published in association with the conference. It reads exactly--"This study provides proof of concept for regenerative medicine approaches of cystic fibrosis using gene corrected adult stem cells upon clonal expansion and selection of stem cells with desired genetic changes. In addition, it indicates that CRISPR-Cas9 dependent approaches in intestinal organoids can be used to create additional human disease models for the study of cystic fibrosis." If you disagree, you are always free to take it up with the investigators at the UMC Utrecht in The Netherlands.

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