Biology 395 24 April 1997 Kenneth L. Campbell

Theodore Friedman, 1989, Progress toward human gene therapy, Science 244:1275-1281.

Modified viral vectors first envisioned in 1970s.

Technical problems first encountered with attempts by Cline et al. 1980 using cloned human beta-globin genes to treat 2 patients with thalassemia.


Gene replacement -- via homologous recombination of the entire gene.

Gene correction -- correction of the mutated sequence in the existing gene, possible via homologous recombination within a gene sequence.

Gene augmentation -- healthy gene replaces the product of a missing or defective gene but does not physically replace the flawed DNA itself.

Transfection often incorporates genes as multiple tandem repeats, some stable, some not.

Transduction via retroviral vectors: low titers in vectors (ca. 1989) a problem, as well as eventual appearance of wild-type virus -- addressed with separate helper virus, specificity of incorporation has been problematic.

Other viral vectors may serve to be tissue selective, e.g., modified herpes virus may allow targeting of nondividing cells such as neurons.

Modified implanted cells for secreted products may be a means of treatment -- problems of reintroduction and maintenance beyond the life of the initial cells.

Inder M. Verma, Gene therapy, Sci Am Nov 1990:68-84.
Somatic vs germ line DNA modification to treat subject only (vs transgenic approaches)

Rare genetic diseases such as Adenosine deaminase (ADA) -- cause of 25% of SCID first targeted.

Augmentation: healthy gene replaces the product of a missing or defective gene but does not physically replace the flawed DNA itself.

Used when no/low protein produced

Doesn't help with overproduction or production of deleterious product (HbS)

Use transduction (viruses: DNA viruses too small or lack latency; RNA retroviruses are preferred) or transfection (chemical means: low efficiency: 1:1000 - 1:100,000 integrates or too slow -- microinjxn) to alter host DNA in subject cells ex vivo.

Retroviruses merge DNA copies only into cells capable of active division -- excludes targets like neurons.

Potential oncogenic agents.

Use of psi- vectors plus helper viruses in packaging system [Diagram]

Use of accessible, robust tissues: bone marrow, skin, liver

ADA/SCID, leukocyte adhesion deficiency, thalassemias among targets.

Treatment of beta thalassemia problematic due to scarcity of erythroid stem cells and need to maintain an optimal subunit balance to maintain a therapeutic effect. But human beta globulin in transduced mouse cells is expressed at low levels; linkage of long range gene-specific enhancers may help. Bone marrow expression also poor.

Ethics of treatment based on positive risk/benefit ratio.

Skin cell fibroblast vehicles: clotting factor IX studies, works but may be limited by immune responses, i.e., unless minimal amounts of protein are made initially by host, it may recognize the therapeutic protein as non-self and mount an immune response to it.

Fibroblasts implants into other tissues like brain may work as vehicles.

Liver cell vehicles: LDL rec has been used as a model system in Watanabe (LDL rec-) rabbits.

Other targets? Endothelial cells, muscle (dystrophin injections), lung (cystic fibrosis treatment via retroviral aerosol)

Other applications: enhancement of disease fighting, TNF insertion into TIL (tumor infiltrating lymphocytes); CD4 production as decoy to HIV

W. French Anderson, Gene therapy, Sci Am Sept 1995:124-128.
First Federally sanctioned therapy: Sept 14, 1990 on 4 yr old Ashanti DeSilva, ADA/SCID. Fully successful with 4 infusions over 4 months (plus several later boosters0 with WBCs transduced ex vivo with modified retrovirus.

Gene therapy = 4th medical revolution (1=public sanitation, 2=surgery+anesthesis, 3=vaccines & antibiotics)

3 approaches: ex vivo, in situ (e.g., Rx of cystic fibrosis, MD via aerosol or injected viruses or DNA, suicide carriers in tumors), or in vivo (nonexistant 1995, e.g., bloodstream injxn with "homing" therapeutic viruses.

Therapy must be viewed in ethical contexts because of potential application to germ line and non-therapeutic situations.

Refer to NIH and Duke documents in notebook, dated late 1995 and after.