State of the Art: GENE THERAPY- Pt2

See part 1

Viral Delivery

Most gene therapy strategies in research and clinical labs up until now have revolved around harnessing the evolved capabilities of viruses to deliver their viral genomes into cells. This is commonly known as use of a viral vector.

Let’s talk a little bit about viruses. Viruses are particles which can infect cells of living organisms. Viruses are made up of a protein shell encasing viral genetic material. In order to reproduce, viruses attach via their protein shells to cell surface membranes where they inject their genetic material. For normal disease causing viruses, the viral genetic material hijacks the cell’s protein and nucleotide generating machinery to produce more complete virus particles. The cycle continues until the immune system can seek and destroy the viral particles (unless the immune system is the target of the virus; as in the case of HIV). The process by which viruses deliver their viral genomes into cells is referred to as viral transduction.

In order to use a virus as a delivery vector, the viral genetic material basically needs to be removed and replaced with genetic material encoding the desired cellular product.

Retroviruses

There are a few different kinds of viruses which can be used for gene transduction. Retroviruses are one kind. Retroviruses store their genetic material in the form of RNA. When a retrovirus infects or transduces a cell, it introduces ins RNA and a few additional enzymes to the cell. The RNA is then copied to DNA inside the cell my an enzyme called reverse transcriptase. The new DNA is then inserted into the cell’s own genome by the integrase enzyme. The viral DNA is now a part of the host cell’s DNA. If the host cell divides, then any daughter cells will share the new DNA. The great thing about that from a gene therapy standpoint is the fact that there would be little or no need to introduce the therapeutic gene more than once. The downsides to it, however, are that:

1) The viral DNA can be incorporated into portions of the cell genome that result in faulty transcription of important genes. This could lead to cancer conditions caused by the gene therapy in the same way that human papillomavirus (HPV) predosiposes women for cervical cancer.

2) If the virus inserts itself into the wrong cell type, the genetic material could be passed on indefinitely within unintended cells for unintended results.

Adenoviruses

Adenoviruses are very different from retroviruses in that the genomic material which adenoviruses use to hijack a cell starts as DNA. Additionally, the DNA does not incorporate itself in the host cell’s genome. The viral DNA finds its way into the host cell’s nucleus where it is transcribed to RNA in the same way all nuclear DNA is transcribed. However, since the viral genes are not incorporated into the cell’s genome, the gene will not be duplicated and passed on to daughter cells after cell division. In one sense, this is advantageous from a gene therapists standpoint. It means that the gene product will only be produced as long as the transduced cells are alive. Long term side effects are minimal. The downside of this approach, however, is the fact that the virus would likely need to be administered more than once.

Adeno-Associated Viruses

Adeno-associated viruses (AAV) are like adenoviruses in that they carry DNA. They are like retroviruses in that the viral genomic material that they carry will be incorporated into the host cell’s genome. Daughter cells will carry the gene, but the genes will not incorporate by integrase into a random portion of the host genome. Instead, AAV always incorporates into chromosome 19. One of the biggest advantages of the AAV, however, has nothing to do with its transduction approach. AAV does not induce an immune response in humans so it can pass through the body as a vector without risk of being destroyed by T cells or macrophages. AAV will not cause fevers or inflammation when administered.

The major downside to AAV is the fact that the viral particles are very small and cannot hold very much genetic material. They would be limited in what gene products they could code for.

So now you know the three types of viruses used for gene therapies. You also know their basic advantages and disadvantages. The next installment in this series will talk about non viral gene delivery techniques. After that, we will summarize the potentially therapeutic gene products being tested in contemporary research labs. We hope you are enjoying the content so far.

State of the Art: GENE THERAPY- Pt 1

What is Gene Therapy?

Gene therapy is the term used for a biological treatment that is designed to introduce new active genetic material to living cells in order to increase or reduce a genetic product or products. These products can include either RNA or proteins or both. For the crudest of analogies, imagine the cell is a factory. This factory has assembly lines that currently build blenders. The blenders are great, but you also want to make toasters now. You send instructions to the factory to reconfigure some of its assembly lines to make toasters for at least part of the time. That is basically what is happening in gene therapy.

The most easily related example that I can think of where this technology could be useful is in Type I diabetes mellitus, where there is a deficiency in production of the protein, insulin, which is encoded by DNA on chromosome 11 in humans. An easy illustration of how a gene therapy could work would be to say that the gene for insulin production could be introduced to cells of a Type 1 diabetes patient so that their body would then be capable of generating insulin on their own. They would no longer need to take insulin shots to control high levels of blood sugar. I will stop there and now posit the emphatic caveat that the case of Type 1 diabetes is much more complex than I just described. The lack of insulin production is not because a gene is missing, rather it is because the cells that normally produce insulin are missing. In fact, diabetes might be better treated with a stem cell therapy than a gene therapy; but I digress (a topic for another State of the Art series). The main point of this ambling monologue is that, by using gene therapy, a new gene or genes can be introduced so that a cell can generate a product that it wasn’t previously generating in order to achieve a variety of net effects.

In the United States, there are currently as many as 30 clinical trials active or enrolling to test the safety and efficacy of myriad gene therapy, or gene transfer, strategies. The trials hope to find treatments for conditions ranging from X-linked Chronic Granulomatous Disease (CGD) where patients’ neutrophils cannot make a key protein necessary for infection defense to Advanced Pancreatic Cancer where a researchers are trying to test safety and dose response of delivering a “tumor killing” gene.

While there are dozens of gene therapy clinical trials and hundreds of labs worldwide conducting research with gene therapy technologies, there is still no FDA approved gene therapy product on the market; nearly 17 years after the first human gene therapy trial was conducted on a 4 year old girl with severe combined immunodeficiency (SCID) at the U.S. National Institutes of Health in 1990.

Why are gene transfers so challenging to develop and administer? There are many pitfalls. First of all, it is simply difficult to incorporate new genes into living cells, especially in a multicellular tissue system. Secondly, once the gene is there, it doesn’t always produce an active protein (or RNA). Thirdly, if the gene does work, it is very difficult, if not impossible, to turn it off, thereby rendering overdoses and immune reactions virtually impossible to treat. Forthly, it is difficult to target genes to show up in the correct cells while not also affecting cells that don’t need the gene. Lastly, there are some questions about potential to pass on the therapeutic gene to offspring who won’t need it.

These issues are currently being addressed with variable success in research around the world. They are testing many different genes and gene deliver strategies in hopes of harnessing biology’s machinery to treat diseases. In the next installments of State of the Art: GENE THERAPY, we will talk about specifics of where the technology is right now. For now, chew on this one. Think of questions. Tell me I am an idiot. Thanks for reading. :)

Upcoming Posts!

State of the Art: GENE THERAPY- Pt2
Viral Delivery

State of the Art: GENE THERAPY- Pt3
Non-Viral Delivery

What did ENCODE decode?

As recently as five days ago, I penned a post about what the Human Genome Project (HGP) had and had not accomplished. I wish I could say that I had written that with the full knowledge that it would be a great primer for a piece about the genome discoveries released today by ENCODE , the NIH follow-up effort to the HGP. I would be lying if I did.

Anyways, front and center at Nature.com is a pdf of the publication by the Encode Consortium outlining the highlights of their efforts to pass a fine toothed comb through approximately 1% of the human genome.

The publication is fascinating in both its breadth and detail. Before I expound on its virtues, let me first comment on my only suspicion about the project. From my own somewhat limited experience in biomedical research, I am not a big fan of large consortium efforts. While I love the concept of open source sharing of data and collaboration, I have usually found that huge efforts across many labs breed data inconsistencies as a result of methodological and analytical differences. Differences in variables as small as humidity in the lab can yield differences in datasets that can obscure the real story. All of that said, it would be very hard to argue with the key points that are coming out of this publication, because the key points make a lot more sense than the conventional wisdom that has been coming out of college biology text books for years (at least when I was in college).

Most of us have been taught at some point that DNA leads to RNA which leads to protein. Well, all of that is still true, but as time goes on, we continue to discover that there are more and more options for the RNA besides producing protein. Without further ado, here are the take home notes on the ENCODE project:

• While it was once thought that a large proportion of DNA was "junk" which did nothing, it is becoming clearer that the vast marority of DNA does transcribe RNA. Many new non-protein coding RNA's have been discovered in the ENCODE effort.
• Chromatin accessibility, basically how tightly the DNA is wound, has a huge effect on how readily it is transcribed to RNA. In turn, many RNA's can affect how tightly the DNA is wound.
• We have evolved in a way that has rendered about 5% of our DNA inactive.
• Some regions of our DNA are wildly variable from person to person, while other regions barely change (this isn't really news, but they've been able to pinpoint some of the specific variable regions).
• RNA can do many things beside encode for protein. Some RNA's are used by the cell to suppress other RNA's...thereby regulating the genome. (this isn't really news either).
  
• There is way too much RNA in cells for us to know what all of it does at this point in time

I do hope you take a look at the pdf file for the original article that I linked to up above. Science journals are tedious to read, especially if it is a new world for you, but it is worth tackling every now and then. There are usually pretty pictures.