Born 30 months ago with severe immunodeficiencydisease (SCID), three small boys are growing up healthy_ little thanks so far to gene therapy.

Rather, the SCID kids, one in Los Angeles, one in SanFrancisco, one in Calgary, Canada, owe their normal livesto regular injections of a bovine enzyme, adenosinedeaminase (PEG-ADA), for which the gene in their ownchromosomes is missing or mutated.

Yet, four days after their birth in late spring of 1993, eachinfant received an implant of normal ADA genes,packaged in a unique delivery vehicle carrying their ownneonatal stem cells.

Stem cells are the little understood, never-seen ancestorsof every mammalian cell in every body. Hematopoieticstem cells, found mainly in blood and bone marrow, areuncommitted and totipotent, like wild cards in poker.

"In newborn infants," observes gene therapist DonaldKohn, "stem cells are baby cells with a whole lifetimeahead of them. During a person's entire life span, theycan give rise to all the different types of blood cells, rederythrocytes, white T lymphocytes, platelets, etc."

In blood and bone marrow, a protein marker of stem cellscalled CD34 can repopulate the entire immune systemdeficient in SCID victims. This is the strategy of ADAgene therapy attempts past, present and future.

The first two successful gene therapy trials in history, justfive years ago last month, introduced cDNA genes intotwo little girls with SCID. (See BioWorld Today, Sept.21, 1995, p. 1.) Michael Blaese, a clinical researcher thenat the National Cancer Institute, with two colleagues, W.French Anderson and Kenneth Culver, convinced theNational Institutes of Health (NIH) Recombinant DNAAdvisory Committee (RAC) and the FDA to permit thisfirst-ever human gene trial.

"In all three little boys we are now treating," Kohnrecalled, "there was a prior sibling or cousin with SCIDin the family. So when the moms conceived again, theyall had amniocentesis to look for ADA deficiency. Andthey found it."

The impending births offered a long-awaited opportunityto Blaese, now at the NIH National Center for HumanGenome Research, and to his colleague, Donald Kohn,who teaches pediatrics and microbiology at the Universityof Southern California.

Belly-Button Blood: Rich Source Of Stem Cells

In every human birth, the final act of delivery is to severthe umbilical cord joining child to mother. This tube ofskin encloses the umbilical artery twined around theumbilical vein.

Umbilical cord blood, which nourishes the fetus duringgestation, should furnish a rich supply of uncommittedstem cells, reasoned Blaese, Kohn and their fellowinvestigators. "Cord blood is baby's peripheral blood,"Kohn explains. "Half of their blood volume at any onetime is out of their body in the placenta. And newborninfants have as many CD34 cells readily accessible intheir blood as adults can provide only by tappingsurgically into their bone marrow."

"We had three months' notice from the time the firstchild was diagnosed in utero, during the winter of `93,"Kohn told BioWorld Today, "to when it was born, onMay 11. Normally," he observed, "it would have taken ayear or two to get permission from RAC and FDA. Butbecause Mike [Blaese] already had that 1990 protocolapproved, we were able to do it by simple amendments."

The blood recovered from each umbilical cord went intoan affinity column, where an antibody harvested all of theCD34 cells. "We got 3, 12 and 18 million of theseneonatal stem cells from the three infants," Kohnrecalled.

The viral vector that infected those cells contained cDNAgene sequences encoding ADA, plus a reporter gene, neo,and a promoter, all strung onto a replication-proof butinfective Moloney retrovirus.

He recalled that four days after the May 11 delivery, "Ipersonally gave the cells to the first patient. A few weekslater, for the second and third infant, we flew them up toSan Francisco and Canada."

Kohn is first author of a paper in the October NatureMedicine titled, "Engraftment of gene-modified umbilicalcord blood cells in neonates with adenosine deaminasedeficiency." Blaese is one of 21 co-authors, from sevenclinical and research centers in the U.S. and Canada.

Transgenes Make ADA In One Cell In 10,000

Over the three boys' first 18 months of life, Kohnestimates, "they probably had only one blood cell in10,000 that carried the transgene." He attributes thisslender level of induction to the inefficiency of retroviralvectors, which can enter only cells that are dividing.

But in those few cells that did take up the ADA genesequence, analysis of the neo selectable drug geneconfirmed expression of natural levels of ADA,"performing the same deamination of adenosine as doesPEG-ADA."

This is the pharmaceutical bovine enzyme on which thethree boys, like all SCID kids these days, survive andflourish.

"The issue with that replacement enzyme," Kohnobserved, "is that it's good for the babies, bad for thegene therapy. All of their good health to this point, wethink, is because of the PEG-ADA. But," he added, "thereason why SCID's been used so much as an early genetherapy model is that we think that the T lymphocytesthat make their own ADA survive without PEG-ADAbeing around."

Early in 1995, with parental consent, Kohn cut the threeboys' PEG-ADA ration in half. "Now," he said, "thenumber of T cells that carry the gene is going up one to10 percent. This implies," he suggested, "that actuallygetting ADA from those genes has given them a selectivesurvival advantage. At this point, eight or nine monthsafter the drop in maintenance dosage, their health remainsthe same." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.