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Early Attempts on Gene Modification

Gene Modification Using Virus as Vehicle

In 1974, a geneticist studied the use of a virus as a vehicle for gene therapy by injecting a gene-modified SV40 virus into early mouse embryonic cells. There were two unexpected findings: (1) Even though the viral genes were present in the cells of the newborn mice, the delivery of the viral genes into sperm and eggs was extremely inefficient; and (2) The expression of the viral genes was completely shut down, resulting in an inert gene that did not make RNA or protein.

After this setback, there were no major advances in gene therapy for a decade, until biologists discovered embryonic stem cells, or ES cells, in 1981.

Gene Modification Using Embryonic Stem Cells

A stem cell is a cell that can (1) give rise to other functional cell types, such as nerve cells or skin cells, through differentiation; and (2) renew itself – i.e., divide to produce more stem cells. Most stem cells live in particular organs and tissues and can only produce special types of cells. Stems cells in the bone marrow, for example, can only produce blood cells. But embryonic stem cells, which live in the inner sheath of an organism’s embryo, are pluripotent, i.e., they can give rise to every cell type in the organism. ES cells also possess some unusual characteristics: (1) they can be isolated from the embryo of an organism and grown in Petrie dishes in the lab; (2) they can be frozen in vials and thawed back to life; (3) the cells can be propagated in liquid broth for generations; and (4) genes can be inserted into their genome or excised from their genome with relative ease.

Using stem cells, a scientist could incorporate a genetically-modified gene permanently into the genome of an animal to form “transgenic” animals. In early 1990s, hundreds of strains of transgenic mice had been created in laboratories around the world to decipher the functions of genes. With gene alterations, one mouse would grow in the dark under blue lamps; others would develop Alzheimer’s disease, epilepsy, or premature aging.  In 2014, researchers created a mouse carrying a mutation in a gene that control the communication between neurons in the brain. These mice have substantially increased memory and superior cognitive function.

Much of the work on ES cells – including transgenic modification of embryos – had been done using mouse cells. Could this technique be used for humans? In the early 1990s, when human ES cells were derived from early human embryos, scientists found out that human ES cells did not behave in culture. “You can’t clone them. You can’t use them for gene targeting…They are very different from mouse ES cells, which can do everything.” So the transgenic modification of human embryos was out of the question for a while.

Gene Therapy Trial for ADA Deficiency

Adenosine deaminase (ADA) deficiency is caused by mutations in the ADA gene, depleting the T-cells, resulting in the collapse of the immune system. The only treatment is to the use of a medicine called PEG-ADA, which has to be injected into the blood every month.

In 1990, a team of gene therapists, led by William Anderson and Michael Blaese, used variants of retroviral vectors to deliver the ADA gene into children with ADA deficiency. The plan was to put the virus into the T cells taken from the blood of ADA patients, and delivered the cells back into the blood of the patients. The T-cells might live just long enough to make the ADA protein and correct the deficiency. Although the T cells would fade from the blood, the procedure could be repeated.

In September, 1990, with the approval of the Recombinant DNA Advisory Committee, Anderson and Blaese performed the gene-therapy trial on two children with ADA deficiency. Did the gene-therapy experiment work? We do not know – and perhaps we will never know, because both patients were allowed continued treatment with PEG-ADA. Any effect of the gene therapy was confounded by that medicine.

Gene Therapy Trial for OTC Deficiency

OTC deficiency is a hereditary disorder caused by the mutation in a single gene involved in metabolism. The mutation causes accumulations of ammonia in the blood, damaging blood vessels and cells, resulting in the slow poisoning of neurons in the brain.

In 1993, two pediatricians in Pennsylvania, Mark Batshaw and James Wilson, experimented with gene therapy to cure children with OTC deficiencies. The form of therapy that Wilson and Batshaw had in mind was more radical than the protocol used by Anderson and Blaese for ADA deficiency. Batshaw and Wilson would create a virus carrying the OTC gene and deliver the virus into the liver through the bloodstream, leaving the virus to infect cells in situ. The virus-infected liver cells would produce the OTC enzyme, correcting the enzyme deficiency.

  In the summer of 1993, Batshaw and Wilson injected the modified adenovirus into mice and monkeys. The mouse experiments worked as predicted, but the monkey experiments were more complicated: some monkeys had liver failure and one monkey died. Wilson and Batshaw changed the virus, and reduced the potential human dose to ensure the safety of the virus. In 1997, they received the approval of the Recombinant DNA Advisory Committee to perform the trial on patients with mild variants of OTC. On the morning of September 13, 1999, Jesse Gelsinger, an eighteen-year-old patient with mild variants of OTC, received the viral injection. Jesse died on the fourth day after the gene delivery.

Investigation into the OTC Trial

A postmortem the OTC trial reveal a “damming pattern of incompetence, blunders, and neglect, compounded by fundamental gaps in knowledge.” An analysis of Jesse’s blood at autopsy found antibodies highly reactive to the virus dating back even before the viral injection. The hyperactive immune response, which was possibly triggered by a prior exposure to a common cold, had spiraled out of control for unknown reasons

In January, 2000, when the FDA inspected twenty-eight other trials, nearly half of them required immediate remedial action. Justifiably alarmed, the FDA shut down nearly all the trials.

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