1) In vivo gene therapy requires that the gene transfer vector be delivered in a cell-type selective manner, either through direct tissue injection, or perhaps someday, by receptor-mediated processes.

2) Ex-vivo gene therapy involves removing tissue from the patient, transfecting (or virally-infecting) the cells in culture, and then reimplanting the genetically altered cells to the patient.


In vivo gene therapy is done by targeting the gene delivery system to the desired cell type in the patient using either physical means such as tissue injection (brain tumor) or biolistics (dermal DNA vaccination), or potentially in the future, using systemic infusion of cell-specific receptor-mediated DNA carriers (reconstructed liposomes or viruses). Importantly, neither of these gene therapy strategies involve reproductive germline cells and therefore the genetic alteration will NOT be transmitted to the next generation. In many countries, human germline gene therapy is considered unethical or even illegal.

Ex-vivo gene therapy is performed by transfecting or infecting patient-derived cells in culture with vector DNA and then reimplanting the transfected cells into the patient. Two types of ex-vivo gene therapies under development are those directed at fibroblasts and hematopoietic stem cells.

Monogenic loss-of-function diseases have been used as paradigms to develop gene therapy strategies using animal models. In this context, gene therapy refers to genotypic alterations (chromosomal or episomal) of somatic cells, resulting in sustained gene expression. This is to be distinguished from transient DNA transfection approaches, such as repeated inhalation or infusion of functional nucleic acids, for example, drug therapies utilizing ribozymes or antisense oligonucleotides.

Each of these gene delivery strategies have their own advantages and disadvantages.

In addition to being safe and cost-effective, the most important properties of an efficacious gene transfer system will be;

1) target cell selective.

2) transcriptionally competent for the desired length of time.

3) available in a highly concentrated active form.

4) immunologically neutral.

While it is still too early to know how useful somatic cell gene therapy will be as a routine treatment for human genetic diseases, most biomedical researchers agree that it will represent just one of the many new molecular genetic tools that 21st century physicians will have at their disposal to better prevent, diagnose and treat their patients.

An example of ex vivo gene therapyis used by Transkaryotic Therapies, Inc., to treat hemophilia A (lack of Factor VIII) and was recently reported in a New England Journal of Medicine article:

Some background on using gene therapy to treat hemophilia A as described in a review that accompanied the Tkt publication in the same issue of the New England Journal of Medicine.

The Tkt primary journal article:
New England Journal of Medicine, June 7, 2001 (vol. 344, pages 1735-1742)
"Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A"
Roth et al. (supported by TKT).

This study was conducted on six individuals with severe hemophilia A all of which received the gene therapy treatment knowingly (open label not double blind). The results showed that a few of the patients did indeed express higher levels of Factor VIII and did not need as much exogenous treatment. However, the effect lasted for less than a year and in several patients there was not much benefit. Let's look at the data:

First off, justification for using this ex vivo gene therapy approach for hemophilia A (factor VIII):

- Factor VIII production is not regulated in response to bleeding

- The broad therapeutic index of factor VIII minimizes risk of overdose

- Delivery of factor VIII into the bloodstream does not require cell-specific expression

- Even low levels of the protein can be beneficial to the patient

The fibroblasts from the skin-biopsy specimen were transfected with a plasmid containing the
gene encoding human factor VIII from which the B domain had been deleted. Prior basic science studies had shown the the B domain is glycosylated and is involved in secretion. Recombinant factor VIII lacking the B domain still functions to stimulate coagulation and has a longer half-life because it is more resistant to protein degradation. The function of the B domain is not known.

Other human gene therapy companies

Cell Genesys in Foster City, California.

Vical Gene Delivery Systems in San Diego, California.

Introgen Therapeutics in Austin, Texas.

Does human gene therapy as it is currently envisioned constitute a form eugenics? Explain.

What limits the number of human diseases that may someday be treated with somatic cell gene therapy?

Can you think of any proprietary information that Tkt may have regarding the actual reagents used in this procedure?


Are there risks for companies like Tkt in putting all of their efforts into ex vivo gene therapy, what would be a good back-up strategy?

Use of human Stem Cells for tissue regeneration and gene therapy

What is a human stem cell? See the NIH primer about stem cells and a more thorough NIH report.

The current state of technology is that much is known about mouse ES cells and how to grow them in culture to "direct" differentiation. With regard to human stem cell technology, it certainly is possible, but it is not yet fully developed to the point where tissue regeneration is possible. Human stem cell research is currently restricted in publicly funded labs (NIH sponsored research at universites) which may signficantly delay progress.

"What is all the fuss about, stems and biology?
Who cares what plant parts are in my salad or in the ziplock baggie I just bought?"
says Rosanne Rosanna Danna.



The basics of Stem Cell Biology

The breakthrough journal article describing the isolation and characterization of human stem cells was published by Science in 1998. The work was done was carried out at the Univerisity of Wisconsin. A non-profit institute called WiCell has been set up at Wisconsin to continue this work, and undoubtedly find patentable applications of the technology. Note that Jennifer Swiergel, one of the co-authors on the original paper earned her PhD here at Arizona with Dr. Gail Burd, good (lucky) choice in post-doc labs, she is now named on the patent!

Science 282:1145 (1998)
Embryonic Stem Cell Lines Derived from Human Blastocysts

James A. Thomson, * Joseph Itskovitz-Eldor, Sander S. Shapiro, Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall, Jeffrey M. Jones

What does the future hold? Much of the near future is in the hands of present adminisration which has limited the use of stem cell technology to a limited number of cell lines, many of which will not be useful. NIH embryonic stem cell registry, click here.


Just for fun, see the quick time movie of stem cell growth in culture.