Introduction to Gene Therapy

Now we're onto the last module for PATH3304: gene therapy!

What is Gene Therapy?

Gene therapy is a series of techniques for solving a defective gene problem in genetic diseases such as muscular dystrophy and sickle-cell anaemia. There are several different approaches that may be used:

• Inserting a normal gene copy into the genome
• Swapping an abnormal gene for a normal gene via homologous recombination
• Repairing the abnormal gene via selective reverse mutation
• Altering gene regulation

The most common approach is the first- inserting a "normal" gene copy. This is usually done through a vector. The most common vectors used are viral vectors, particularly adenoviruses (dsDNA), adeno-associated viruses (ssDNA that can insert their genetic material at a specific spot on chromosome 19), HSV (dsDNA) and retroviruses (can create double-stranded DNA copies of their RNA genomes). Viral vectors do not always need to be used: DNA can also be transferred by direct injection, electroporation, gene guns, liposomal DNA and whole-cell transplantation.

X-SCID

X-SCID, or X-linked severe combined immunodeficiency, can be potentially treated via gene therapy. Bone marrow cells are isolated from the patient and transfected in vitro with recombinant retrovirus with "normal" versions of the defective genes. After testing to ensure that the bone marrow cells have taken up the normal gene, the cells are returned to the patient.

Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD), as also discussed here, is an X-linked recessive disorder in which there is dysfunctional dystrophin. Dystrophin is a 427 kDa protein with an actin-binding domain at one end, rod domains and hinge regions in the middle, and a cysteine-rich binding region and C-terminal domain at the other end. In DMD, there are often nonsense mutations causing the formation of a truncated dystrophin protein, or mutations in the binding domains that prevent dystrophin from carrying out its normal role. In BMD (Becker Muscular Dystrophy), which is a less severe form of the disease, these mutations usually affect the rod domains, resulting in a shorter but still functional form of dystrophin.

Several genetic therapies have been proposed for treatment of DMD:

• Mini dystrophin gene delivery: Since the normal dystrophin gene is too large to put into a plasmid or viral vector, delivery of a BMD-like dystrophin gene has been suggested. As I just mentioned, BMD dystrophin is shorter than normal, but at least it's still functional.
• Transcriptional read through: Some antibiotics will cause ribosomes to ignore stop codons, which would stop the formation of truncated proteins.
• Exon skipping: Alternative splicing to cut out some of the mutated regions. The idea is that this will hopefully result in a shorter but still functional form of dystrophin.
• Homologue induction: Embryos use a homologue called utrophin instead of dystrophin. Perhaps utrophin could be selectively upregulated to make up for the lack of dystrophin.
• Cell-based whole gene replacement: Transplantation of donor muscle precursor cells (myoblasts) that have functional dystrophin genes.

In this post, we will be focusing mainly on cell-based whole gene replacement, which in this case is called MTT (Myoblast Transfer Therapy). In MTT, dystrophin-expressing donor myoblasts are injected into dystrophic muscle. Unfortunately, myoblast survival following transplantation is very poor, possibly due to the effects of the acquired immune system.

In order to combat the acquired immune system response, researchers have looked at Tregs (regulatory T-cells), which are CD4+ CD25+ T-cells that can suppress the immune response. They release anti-inflammatory cytokines, such as IL-10 and TGFβ. IL-10 and TGFβ can also be expressed by plasmids, such as pMP6a. pMP6a has an ampicillin resistance gene, a cytomegalovirus promoter, and unique restriction sites that can be digested with Nhe1. pMP6a can be taken up by E. coli by electroporation, allowing E. coli to act as little factories for making more and more pMP6a with the genes of interest. Since pMP6a encodes ampicillin resistance, E. coli cultures that took up the plasmid can be selected for by culturing with ampicillin.

Once lots of pMP6a plasmids have been produced, we need to test that they can be taken up by eukaryotic cells, such as Cos-7 cells. ELISA can then be used to ensure that Cos-7 cells are indeed producing the cytokines of interest. Once that has been confirmed, injection of plasmid and in vivo electroporation can be used to deliver cytokines to the muscle. Studies in mice have shown that this technique does increase levels of anti-inflammatory cytokines, as anticipated. Unfortunately, even though there was less inflammation, this didn't really help with the survival of transplanted myoblasts. Oh well, back to the drawing board I guess!