Gene Therapy III

• retroviruses as vectors (see Gene Therapy I)
• adeno-associated virus (AAV) as the vector (see Gene Therapy II)

One problem with these approaches is that foreign DNA is inserted into the host genome. It is possible — and has been demonstrated — that the foreign DNA may be inserted into a chromosomal position that disturbs normal gene function there. In fact, several boys treated with vectors containing a gene to cure their X-linked severe combined immunodeficiency (X-linked SCID) developed cancer because of this. [Link]

But now Urnov, F. D. et al., report (in Nature, 6 June 2005) their success — with cultured cells — in correcting the molecular deficiency in X-linked SCID without the need for any vector.

X-linked SCID is caused by a mutated X-linked gene encoding a subunit — called γc (gamma-c) — of the receptor for several interleukins.

1. a zinc-finger transcription factor. This can be engineered to recognize and bind to any desired DNA sequence in the genome. It is coupled to
2. a restriction enzyme that cuts through both strands of DNA near that location. The combination of 1 and 2 is a "zinc-finger nuclease".
3. a separate plasmid containing the correct version of the γc gene.

The result: a double-stranded break (DSB) in the DNA at the γc locus.

• their own machinery for the repair of DSBs by homologous recombination and
• the plasmid as a template

• not only avoids the danger of introducing DNA into random sites in the genome but also
• avoids the need for introducing foreign genes to aid in selection of successfully-repaired cells [Link to discussion].

It's a long way from something that works in cultured cells to something that works in human patients, but here at least is a promising procedure. Instead of adding a functioning gene anywhere in the genome, both copies of the cell's own defective genes are repaired.

Humans with single-gene disorders like

• sickle-cell disease
• severe-combined immunodeficiency (SCID)

• removed
• treated in vitro by this method and then
• returned to them.

But what of genetic diseases whose gene products are produced by immobile cells in organs like the liver?

• Li, H. et al reported in the 14 July 2011 issue of Nature that they have made the jump from repairing cultured cells in vitro to restoring gene function by treating intact animals. They succeeded in curing mice of hemophilia B by injecting the livers of mice whose own genes for the blood clotting factor 9 (IX) had been deleted and replaced with a mutated, non-functional, human factor 9 gene. The injected vector contained
  • zinc-finger nucleases targeting the first intron of the mutated factor 9 gene causing a double-stranded break (DSB) there;
  • a functional factor 9 cDNA to serve as a template for repairing the disabled gene.

  These treated mice began to secrete low levels of human factor 9, and their blood began to clot normally.

• Yusa, K. et al reported in the 20 October 2011 issue of Nature that they
  • produced induced pluripotent stem cells (iPSCs) from humans with alpha1-antitrypsin deficiency;
  • transformed these with the unmutated human gene — using zinc-finger nuclease technology;
  • caused these to differentiate into liver cells which secreted functional alpha1-antitrypsin.
  • When transplanted into immune-deficient mice, these human cells took up residence in the liver where they continued to synthesize and secrete human proteins, including alpha1-antitrypsin, for 5 weeks.

  We are now a step closer to correcting human single-gene diseases with patient-specific cell transplants!