Gene Therapy thoughts

As I understand the procedure for the gene therapy is as following:

General process for of the most gene therapy studies is:

1.    "Normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene.

2.    A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells.

3.    Target cells such as the patient's liver or lung cells are infected with the viral vector.

4.    New functional protein product from the therapeutic gene restores the target cell to a normal state.

Applying this process to our specific needs:  

1.    AGA gene is known and very well defined in the literature. There are a lot of data available. I put some more info on the website.

The gene is 11 734 bp long, containing 9 exons. It’s been reported that mutations occur in all 9 exons, but 5^th; so we should target at replacing the whole gene to cover all the variations of AGU disease.

Cells (RNA) copy only the information carried by exons to make proteins. So, the length of the DNA material needed to make AGA enzyme is about 255+154+113+113+115+76+108+134+1039=2107 bp.  It is a very good news!!!

2.    There are different types of viruses used as gene therapy vectors. Mostly commonly the following viruses are used:  retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses. The reported drawback of the viruses are:

a.    Short-lived nature of gene therapy

b.    Immune response

c.    Problems with viral vectors

d.    Multigene disorders – not an issue in our case, single gene, AGA

For the first three issues, one particular virus can solve them. It is adeno-associated virus (AAV).

AAVs are a class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. It inserts into genetic material, so will live for a long time in a body and only single injection might be needed. It has no reported immune response problems. You can read more general information about it in English and a little in French (sorry).

We probably will be interested in working with AAV type 2. Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells (that exactly what we need!) and Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. (we like that one, too!)

The reported problem with AAV is the small size, which is about 5000 bp, meaning that the size of gene/molecule that AAV can carry must be less than 5000 bp. It is a small molecule and a lot of genes are much larger.

That is where the size of AGA exons comes. The size of genetic material required to make a good AGA protein/enzyme is only 2107 bp. So, it should fit into AAV!!!!! There are methods to cut a gene to exons and make a smaller gene, something that called plasmid DNA and cDNA. Here is an example of an 11000 bp gene that they were able to put into AAV. There are also companies in USA that make plasmid DNA commercially.  

3.    Before human trials, mouse and human skin fibroblast trials should be done. We need to consult and figure out the best place to do it from approval/legal point of view.

Also, I found the article that talk about gene therapy for AGU work done in Finland and France in 1998 already. I don’t think they finished the work. The results were very good! You can download the full article through the link. (maybe just wrong vector-carrier, so we’ll fix it)

4.    And we wait and see the results!

Let me know your ideas and thought. JL