Introns, exons, and stop codons: how antisense oligonucleotides can fix frameshift mutations

DMD is the largest gene in nature, covering roughly 2.4 mega bases of the X chromosome. It encodes the dystrophin protein, a component of the protein complex that connects the cytoskeleton to the extra-cellular matrix.

DMD is a very complex gene. Its RNA transcripts are differentially spliced, which means that the gene produces different transcripts, encoding a large set of protein isoforms. A refresher: every gene is composed of coding parts, called exons, interspersed with non-coding bits, called introns. When the gene is transcribed into RNA, a process called RNA splicing, the introns are removed and the exons (grr… my auto-correct keeps turning all my "exons" into "eons"!) reassembled to form the RNA transcript that will be used to form proteins. Some proteins, like dystrophin, have different isoforms (some specific to different cell types), and those are obtained through different splicing forms of the RNA, originated by maintaining a different number of exons in the final transcript.

This video is a good illustration of RNA splicing:

All this to give you an idea of how complex this gene is. So, when it carries a mutation, things get very complicated, and the consequences devastating. Mutations in the DMD gene are responsible for several forms of muscular dystrophy (MD), and because the gene is on the X chromosomes, the prevalence is usually higher in boys than girls. (This is because girls carry two X chromosomes, hence if one allele only is mutated, the other will compensate.)

The most common mutations causing muscular dystrophy cause the transcription process to stop too early, producing incomplete, and therefore non-functional, RNA transcripts. I discussed reading frames in this post: in layman terms, the reading frame of a gene is how you split the bases in triplets so that each triplet codes one amino acid (the building blocks of proteins). Mutations that cause a shift in the reading frame basically disrupt the translation into amino acid, often resulting in the early termination of the transcription process (when the frameshift causes the random appearance of an early stop codon). When not enough functional dystrophin is produced, individuals experience a significant loss in muscle function and muscle degeneration.

How to counteract the action of frameshifting mutations?

One way is to use antisense oligonucleotides, buts of RNA that bind to a splicing site on the pre-mRNA causing the deleterious exons to be skipped and thus restoring the "functional frame." What does this mean? Remember, RNA is one-stranded. From DNA to RNA there are several steps: pre-messenger RNA and messenger RNA, or mRNA. The deleterious mutations are on the gene and they cause a misread when going from DNA to pre-mRNA. Now the mutations are on the pre-mRNA. Suppose you can devise a "bandage" that literally covers the bit of bases causing the framshift. If the bandage works, the bit won't be read when the pre-mRNA is turned into mRNA thus effectively canceling the frameshift and restoring the original RNA transcript. These "bandages" are bits of antisense RNA specifically made to bind to the "bad" parts of pre-mRNA. I covered this kind of therapy in an earlier post on gene therapy.

Does it work? So far, enough to give hope.

Goemans et al. [1] recruited 12 patients with Duchenne's muscular distrophy. Over the course of 12 weeks, the patients received weekly, dose-escalating

"weekly abdominal subcutaneous injections of PRO051 (from 0.5 to 10 mg per kilogram of body weight, with 3 patients receiving each dose) for 5 weeks. The specific increases in dose were determined after analysis of safety and dystrophin levels in muscle-biopsy specimens."

The lowest dose of 0.5 mg per kilogram showed no effect on RNA or protein expression. Exon-skipping RNA was instead observed in the higher-dose patients. Muscle biopsies were sampled at the end of the high-dose period and new dystrophin expression was observed starting from week 2, with increased signal as time and dose progressed. By the end of the twelve weeks the average distance walked in six minutes across all patients had increased by 35 meters, with some patients able to walk 65 meters farther than at baseline. Patients were also tested 2 and 7 weeks after the treatment, and most still showed similar dystrophin expression levels as right after the treatment.

Though a lot still needs to be done in order to defeat this disease, these results certainly set a much needed step forward.

[1] Goemans, N., Tulinius, M., van den Akker, J., Burm, B., Ekhart, P., Heuvelmans, N., Holling, T., Janson, A., Platenburg, G., Sipkens, J., Sitsen, J., Aartsma-Rus, A., van Ommen, G., Buyse, G., Darin, N., Verschuuren, J., Campion, G., de Kimpe, S., & van Deutekom, J. (2011). Systemic Administration of PRO051 in Duchenne's Muscular Dystrophy New England Journal of Medicine, 364 (16), 1513-1522 DOI: 10.1056/NEJMoa1011367

This picture was a "Rule of Thirds" exercise: find an interesting background, and have the main subject of your photo cover one third of the picture only, instead of positioning it in the middle. You can see how it immediately makes both subject and background more interesting. Focal length 26mm, shutter speed 1/25, ISO speed 100, F-stop 7.1

Sense and antisense in the human genome

I hope you all had a wonderful holiday. Short post today, as I'm sure we're all still digesting all the yummy holiday food and sweets, and maybe some of you are still celebrating. One of my recurrent topics on the blog has been antisense genes. Until recently, I had no idea such things existed, let alone in humans. It turns out, they are quite abundant in humans.

Antisense genes are overlapping genes that are transcribed on opposite DNA strands. I've discussed how antisense genes regulate conjugation in bacteria, and how antisense RNA transcripts can be used in gene therapy. Today I'd like to discuss a paper that examined five different human cell types and found evidence for antisense transcripts in thousands of genes.

As you know, a gene is a piece of DNA, and a gene transcript is the RNA trasncribed from that gene. DNA is made of two strands coiled together, which are conventionally referred to as the plus strand and the minus strand. The general thought has been that sense transcripts produce functional proteins, whereas antisense transcripts have regulatory functions. For example, they can "silence" a gene since the antisense RNA will attach to the sense RNA and a double-stranded RNA can no longer produce a protein.

In [1], He et al. developed a technique that allows to change the RNA transcript in a way that, once turned back into DNA, it will only match either the plus or the minus DNA strand. This way one can establish from which strand it had been transcribed. The researchers analyzed five cell types: PBMC, peripheral blood mononuclear cells isolated from a healthy volunteer; Jurkat, a T cell leukemia line; HCT116, a colorectal cancer cell line; MiaPaCa2, a pancreatic cancer line; MRC5, a fibroblast cell line derived from normal lung. They called "S genes" the ones that contained only sense tags or had a sense/antisense tag ratio of 5 or more; "AS genes" contained only antisense tags or had a sense/antisense tag ratio of 0.2 or less; and finally, "SAS genes" contained both sense and antisense tags and had a sense/antisense ratio between 0.2 and 5.

I found this figure in particular to be quite interesting:

From the figure, it's clear that sense genes tend to accumulate in the exons (the coding bits of a gene), whereas the antisense genes accumulate more in the promoters, regions upstream of a gene that regulate and promote transcription, and, though to a less extent, in the terminator regions. The authors of the paper used these data to argue that, while

"promiscuous expression would lead to a uniform distribution of antisense tags across the genome, the observed distribution was nonrandom, localized to genes and within particular regions of genes, much like sense transcripts."

In other words, antisense genes are non-randomly distributed and may in fact contribute to antisense-mediated regulatory mechanism that, according to the data presented in [1] affects from 2900 to 6400 human genes. More on this in the next post! Happy Holidays, everyone!

[1] He, Y., Vogelstein, B., Velculescu, V., Papadopoulos, N., & Kinzler, K. (2008). The Antisense Transcriptomes of Human Cells Science, 322 (5909), 1855-1857 DOI: 10.1126/science.1163853

A battle for transcription regulates bacterial conjugation

Genetic information is transmitted in two modes: when we talk about the slow accumulation of mutations across generations, we are talking about vertical gene transfer, in other words, the transmission of genetic alleles from the parents to the offsprings. Genetic material can also be transferred "horizontally" when an organism incorporates another individual's genetic material without being the individual's offspring. A genetic chimera is an example of a horizontal gene transfer.

You can picture horizontal gene transfer as a sudden increase in genetic diversity. While most of evolution studies have focused on vertical gene transfers, horizontal gene transfer has been shown in some milestones in the evolution of life: for example mitochondria have originated through a horizontal transfer event from an eukaryotic cell which incorporated a bacteria by symbiosis.

Bacterial conjugation is the horizontal gene transfer process through which bacteria exchange genetic material. That's what makes bacteria so efficient at developing antibiotic resistance. It takes many mutations to find the ones that confer resistance, but once the mutation appears in the population, it spreads to other individuals very quickly. How?

Besides the usual strand of chromosomal DNA, bacteria have a separate DNA molecule called plasmid, which is a short bit of double-stranded, circular DNA (circularity makes it more stable). The plasmid is what gets transferred during bacterial conjugation. The process involves a donor cell and a recipient cell. The donor has the plasmid with the gene that confers antibiotic resistance, and the recipient doesn't. Once the donor "recognizes" that the nearby cell lacks the resistance gene, a channel gets opened from the donor cell to the recipient. One of the two DNA strands in the plasmid is cut, unrolled, and transferred to the donor cell through the channel. Both cells then produce the complementary strand, and the original plasmid is restored in both.

But how does the donor cell know that the nearby cell does not have the resistance gene? Each cell communicates by expressing different peptides and "sensing" the neighbor's peptides through a mechanism called "Type 4 secretion system," or TFSS. Once it "detects" that the neighbor doesn't have the resistant gene, conjugation is activated.

Chatterjee et al. [1] studied the mechanism in Enterococcus faecalis and presented a mathematical model (supported by experimental data) of conjugative transfer regulated through convergent transcription from antagonistic genes, in other words, genes that sit on opposite strands of the DNA and hence compete for transcription.

Two genes on the plasmid regulate conjugation: gene Q activates it, and gene X represses it. Now, here's the interesting bit: X and Q are overlapping, sense-antisense genes. This means that they get transcribed in opposite directions. Remember: transcription is the process that converts DNA into a single strand of RNA, which is what the cell needs in order to produce proteins. Think of the single stranded RNA as a list of instructions that needs to get through. If the RNA from gene Q is produced, then conjugation is activated and the plasmid transfer occurs. If RNA from the X gene is produced instead, conjugation is repressed.

RNA transcription is carried out through an enzyme that "slides" through the DNA much like a zipper. The novel idea of this paper is that if the genes are transcribed in opposite directions, the two enzymes transcribing each gene will "collide" with a certain probability. One enzyme slides in one direction, the other in the opposite direction, and depending on how frequently the process takes place, the two enzymes "crash", interrupting the transcription process, as illustrated in the graphics below (by Kaitlyn Pladson and Ranja Sem).

Each time a collision happens, incomplete strands of complementary RNA are created. Complementary RNA strands will "stick" together and when that happens they can no longer be used to make proteins. As a result, the two enzymes are effectively competing against one another for which of the two genes gets transcribed: where and how frequently they collide regulates the activation of either the gene X or the gene Q, thus initiating or repressing bacterial conjugation. This kind of competition between the two enzymes due to the relative expressions of sense and antisense genes is what regulates the switch between activating the conjugation or repressing it. In the end, the enzyme that is able to zip through the gene faster ultimately "wins" because it is able to produce a larger concentration of RNA strands.

As I read the paper, I couldn't help but wonder how many other biological mechanisms are regulated by this sense-antisense antagonistic transcription. We know there are sense-antisense genes in the human genome, and little is known about their function. This study sheds new light into these DNA regions and advocates for more equivalent research in the human genome. As Chatterjee et al. conclude in their paper, "The fact that convergent transcription is ubiquitous and has persisted in evolution is perhaps an indication that such gene organizations confer fundamental mechanisms of gene regulation. With such a wide range of possible outcomes, using subtle structural tuning, convergent transcription may be highly adaptable to become a robust controller for many complex cellular events."

[1] Chatterjee A, Johnson CM, Shu CC, Kaznessis YN, Ramkrishna D, Dunny GM, & Hu WS (2011). Convergent transcription confers a bistable switch in Enterococcus faecalis conjugation. Proceedings of the National Academy of Sciences of the United States of America, 108 (23), 9721-6 PMID:
21606359

Photo: morning glories. Canon 40D, shutter speed 1/400, focal length 85mm, f-stop 5.6, ISO 100.

Gene therapy makes sense. And antisense, too!

Genes code proteins. When a gene carries a defective mutation, it will either stop coding the protein or it will code a defective one. This is, unfortunately, the basis of many genetic diseases. In principle, if we could substitute the defective gene with a healthy one, the problem would be solved. That's what gene therapy boils down to. In fact, there are ways to deliver the genes to the affected cells. For example, you can take a virus that targets the cells where the defective gene is expressed, keep the virus's ability to inject its genome into the target cell, but modify its genetic content so that now it contains the healthy genes. The virus will "attack" the target cell in its usual manner and inject its genetic content inside. But now, because the virus has been artificially modified, the new genetic content won't be the usual viral genes that cause infections. Instead, they will be the new, healthy genes, which will be integrated in the cell's DNA and start coding the healthy protein.

Gene therapy has been used successfully to treat various genetic diseases (see the studies listed here) and there have been very encouraging results when used to treat cancer in mouse models (see, for example, [1]) as well as in humans [2].

However, there are situations where the "simple" scenario I described above will not work. 

We are diploid organisms, which means we carry two copies of each chromosome, and hence two copies of each gene. The two copies may or may not be identical. When they differ, we say that the individual is heterozygous at that particular locus. Now, it so happens that in some heterozygous individuals one of the two copies of the gene is dominant negative. What that means is that even if the other copy is healthy, and it codes a healthy protein, the defective protein (produced by the defective gene) interacts with it and alters its function. Basically, the mutated protein dominates over the non-mutated one and overrides its ability to function properly.

When this happens, "delivering" the healthy gene will not solve the problem because the defective gene will continue to produce the defective protein, which, in turn, will override the effect of the healthy one. Does this mean we can't use gene therapy to fix the defective gene? Of course we can! We just have to use a different kind of gene therapy, namely antisense gene therapy.

This is how it works.

I've used the expression "genes produce proteins." Well, it's a little more complicated than that. Each strand of DNA gets first transcribed in RNA and then the RNA (which is single stranded) is translated into the protein. The idea behind antisense gene therapy is to prevent the defective RNA strand to be translated into the defective protein. How? By binding the defective RNA before it can be used by the cell to make the defective protein.

DNA is made of two strands that are complementary to each other. The same principle works for RNA, even if RNA is usually found in single strands. So, if you make its complementary (the antisense strand), it will bind to it like opposite magnets do. And that's exactly what antisense gene therapy does: instead of delivering pieces of DNA, it delivers pieces of antisense RNA made to complement exactly the defective RNA.

The figure below is from this website:

As you can see from the figure, the defective RNA is "plugged" by its antisense complement and at that point is no longer able to make the defective protein. The healthy protein, produced by the unmutated copy of the gene, completely takes over thus eliminating the source of the disease.

References [3] and [4] below show examples of antisense gene therapy used in treating brain and cervical cancers. 

[1]  Suto, R., Tominaga, K., Mizuguchi, H., Sasaki, E., Higuchi, K., Kim, S., Iwao, H., & Arakawa, T. (2004). Dominant-negative mutant of c-Jun gene transfer: a novel therapeutic strategy for colorectal cancer Gene Therapy, 11 (2), 187-193 DOI: 10.1038/sj.gt.3302158

[2] Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, & Rosenberg SA (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science (New York, N.Y.), 314 (5796), 126-9 PMID: 16946036

[3] Zhang Y, Zhu C, & Pardridge WM (2002). Antisense gene therapy of brain cancer with an artificial virus gene delivery system. Molecular therapy : the journal of the American Society of Gene Therapy, 6 (1), 67-72 PMID: 12095305

[4] Yatabe, N., Kyo, S., Kondo, S., Kanaya, T., Wang, Z., Maida, Y., Takakura, M., Nakamura, M., Tanaka, M., & Inoue, M. (2002). 2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells Cancer Gene Therapy, 9 (7), 624-630 DOI: 10.1038/sj.cgt.7700479