Minutes - November 10, 1998 : NIDDK

Minutes - November 10, 1998

Member attendees: David Badman, NIDDK; Harish Dave, VAMC (alternate for Dr. Patel); Allan Lock, NICHHD; Helena Mishoe, NHLBI; Helen Quill, NIAID; Charles Rodgers, NIDDK; Grace Shen, NCI; Dorothy Sugn, NCRR (alternate for Dr. Wilde)

Guest attendees: Libin Jia, NHGRI, MGA; E. Schroder, NIAID; Jennifer Fain-Thornton, NIDDK; Jianwen Wu, NIDDK; Vladimir Ponomarev, CNMC; Martha Liggett, American Society of Hematology; Gang Zeng, NCI; James Chan, NIDCR; D. Bodine, NHGRI; Greg Evans, NHLBI; Charles Peterson, NHLBI; Richard Krazek, NCRR; Caroline Lee, NCI

Guest speaker: Dr. W. French Anderson

Dr. David Badman welcomed attendees to this meeting of the Hematology Subcommittee and introduced the guest speaker, Dr. W. French Anderson, Director of the Gene Therapy Laboratories, Norris Cancer Center, University of Southern California School of Medicine.

Dr. Anderson outlined the topics of his discussion: his new work involving murine stem cells and the application of this work to gene therapy using retroviral vectors, new technology that has resulted in surprising findings; and in-utero gene therapy proposals.

Initial clinical trials of gene therapy, he noted, have not been very successful, due in part to the natural human defense system against foreign DNA. Translating in vitro results into in vivo success has therefore been very limited. At the first meeting in May 1998 of the American Society of Gene Therapy in Seattle, Washington, however, researchers who had examined these early failures in an attempt to learn from past mistakes shared their sense of excitement about preliminary success with their new approaches.

Dr. Anderson introduced the basic mechanics of cell lineage maturation, from pluripotent stem cells to lymphoid and myeloid stem cells to mature blood cells. At present, researchers study stem cells as a population of cells, not as specific cells. Dr. Anderson defined the pluripotent hematopoietic stem cell (PHSC) as having in vivo capacity for long-term repopulation for all blood cell lineages in vivo. The PHSC can be studied in a number of ways, but Dr. Anderson has concentrated his research on cell surface markers in the hope of expanding the knowledge of PHSC subsets.

Studies released in 1996, including one by a Japanese group, used four different antibodies to identify cells: antibodies specific for Lin, CD34 Sca Kit. The cell Lin(lo)Sca-1(+)Kit(+)CD34(-) was suggested as the earliest PHSC. A second study (Randall and Weissman) examined CD38 rather than CD34, which is likely to be different between human and mouse. These were both four-color studies; Dr. Anderson has succeeded in conducting a five-color study, examining CD34 and CD38 in the same stem cell population. A brief outline of this study follows.

Dr. Anderson conducted competitive repopulation assays using donor cells (n=10, 45, or 100) from C57BL/Ly5.1 and competitor whole bone marrow cells C57BL/Ly5.2 [2 to 4 X 105]. When the donor cells are placed into an irradiated mouse that is protected by C57BL/Ly5.2 cells, one can examine the functioning of the Ly5.1 cells. In addition, he used the CFU-S assay, which looks at a window just after the earliest stem cell and just before the cell differentiates.

The lineage negative cells were gated in the FACS against forward and side scatter and then gated against Sca and Kit. The mid-intensity cells were intentionally left out of the study, so positives were true positives and negatives were true negatives. The Lin(-)Sca(+)Kit(+) subset was gated against 34 and 38, leaving four subsets of stem cells that were examined: Lin(-) Sca(+)Kit(+)34(+)38(+), abbreviated as ++++; Lin(-)Sca(+)Kit(+)34(+)38(-), abbreviated as +++-; and Lin(-)Sca(+)Kit(+)34(-)38(+), abbreviated as ++-+. ++-- were too few in number to study. These subsets were combined, to compare results with similar previous studies, and separated, to discover new data on stem cell maturation. These three populations were examined to determine how these cells behave during long-term repopulation of irradiated animals.

Conclusions: +++- cells have both long-term and short-term repopulating abilities; ++++ and ++-+ have short-term, but no long-term, repopulating ability; therefore, +++- are the more primitive PHSCs. The ++++ and ++-+ contain at least some CFU-S12 but +++- cells do not. Possible order of maturation (from least to most) is, therefore, +++- to ++++ to ++-+. The 34(+) cells are only short-term repopulating, whereas 34(-) are long-term repopulating cells.

At this point in his presentation, Dr. Anderson answered a question on the morphology of the cells, clarifying that the cells appear to have identical morphologies but have different in vitro and in vivo properties.

Short-term repopulating cells (STRC) will protect the animal from lethal irradiation for a matter of weeks; long-term repopulating cells (LTRC) are thought not to protect the animal in the short term but do protect the animal in the longer term (starting at 3 months and continuing through the lifespan of the animal).

Dr. Anderson noted previous research in this area (Jones et al., Nature 347:188-9, 1990; and Osawa et al., Science 273:241-5, 1996).

Conclusions (continued): At 5 and 16 weeks, all three populations repopulate. Not only do the +++- cells provide long-term repopulation, but they also protect in the short term better than the STRC. This is the one area where Dr. Anderson's research provides data contrary to the literature. He carried out the experiments at days 3, 8, 14, and 21 to determine the earliest onset of LTRC. The earliest the LTRC were found in the bone marrow was 3 days. At 2 weeks they showed up in peripheral blood, and by 5 weeks they were outgrowing the other populations. These other populations slowly decreased in size until they could not be found at 8 months.

Dr. Anderson noted these unresolved issues: At present, three labs have shown that CD34(-) appear to be more primitive in the mouse, and this finding may also be true in humans. In humans, CD34(-) appear to give long-term repopulation and mature into CD34(+), even though the CD34(+) have been shown to have long-term repopulation ability. In the monkey CD34(-) cells appear to be more primitive; perhaps these results will prove to be analogous to human stem cell development.

Turning to the problem of placing genes in stem cells and in-utero gene therapy, Dr. Anderson posed the question, "Why has gene therapy not been working well?" Drawing on his review in the April issue of Nature, he offered two reasons. First, we cannot get genes into a sufficient number of cells in the patient. Second, even if one successfully solves the first problem, the gene usually gets turned off. In general, we are missing an effective delivery system and effective vectors that can give stable, long-term expression.

When treating blood or genetic diseases, the hematopoietic stem cell would be the best cell to target. How can we alleviate the delivery and expression problems? Expression is complicated because the human body defends itself. A human cell in vivo can recognize, in ways that are not understood, if the gene is being controlled by normal, authentic human control regions or by viral control regions. When a viral control region is recognized, the human body appears to be able to shut the gene down by methylation or other means.

By using authentic human genomic regulatory sequences, Dr. Anderson hopes to bypass this obstacle. However, this too has problems. We know the regulatory sequences for only a few genes, and these sequences are extremely large, most covering tens of kilobases.

Dr. Anderson chose to concentrate on two diseases, ADA (adenosine deaminase) deficiency and alpha-thalassemia, both of which contain well understood human control regions. Bruce Aronow at Cincinnati has spent many years looking at the human control regions that regulate ADA in human T-cells, working out the sequences and cutting them down. He placed plasmids in transgenic mice and demonstrated that he could get the appropriate regulation of ADA in T-cells in the mouse using his truncated human control regions.

The first disease Dr. Anderson has chosen for developing a new generation of vectors is ADA SCID (severe combined immune deficiency). He is deleting the viral regulatory sequences and using only human sequences. The approach will use a direct injection of the retroviral vector into the peritoneal cavity of the 13-15 week-old fetus. The fetus was chosen as the experimental patient for ADA gene therapy with the goal of a born child that requires no further therapy. The pre-protocol proposal was presented to the NIH RAC about a month and a half ago. To obtain a copy of this proposal, one can call (301) 496-9838 and request the mailings for the September RAC meeting.

Dr. Anderson reviewed data from his first ADA gene therapy experiment. Initially after receiving PEG-ADA, the patient had a significant response but, unfortunately, her T-cell levels then began to gradually decrease. Once started on gene therapy, however, after a few initial problems, her T-cells rose and remained normal. Since August of 1992, the patient has received no additional monthly injections of the corrected T-cells but has remained on PEG-ADA. The data demonstrate that the child's health improvement is the result of the gene-corrected cells, but she remains on PEG-ADA as a precautionary measure. Why give monthly injections of the gene-corrected cells (a total of 11 over the 23 months)? The infusions were administered bi-monthly to mimic the actions of the naturally variable human immune system with a repertoire of T-cells that change on a daily basis.

The initial fluctuations in T-cells was not reflective of the corrected cells; it appears that by having sufficient gene-corrected cells in the lymphoid spaces, uncorrected cells are able to divide and function normally. Over the past six years, 75 percent of the patient's T-cells are uncorrected T-cells, but they appear to be functioning relatively normally.

In ADA deficiency, the corrected cells have a positive growth advantage: a corrected cell divides normally, but a defective cell cannot divide. This is not the case in other common diseases, namely, cancer, heart disease, many other genetic diseases, arthritis, and AIDS. Gene therapy will not work as successfully as it does with ADA in cases where there is no positive growth advantage for the corrected cells.

The second disease Dr. Anderson is focusing on, homozygous alpha-thalassemia, produces a particularly horrible disease that kills the fetus in utero, making the dying fetus toxic to the mother. There is currently no therapy for the fetus, and the only course for the protection of the mother is an abortion at 24 weeks. Previous studies in sheep by Dr. Anderson and Dr. Esmail Zanjani, published in 1989-1990, outlined a procedure involving removing the autologous blood stem cells from the fetus, ex vivo putting in a gene, and injecting those gene-engineered cells into the fetus at 17-20 weeks of gestation. This protocol will be used in the study proposed to the NIH-RAC. Human hemoglobin regulatory sequences will be used in this study.

Following a question and answer session, Dr. Badman thanked Dr. Anderson for his lecture, excused all guests, and began the business part of the meeting.

Dr. Grace Shen from NCI distributed printouts from the NCI Web site and briefly summarized her agency's work on developing reagents and a centralized database to help identify new cancer chromosomal aberrations. The agency plans to extend this effort to include genomic reagents across the entire genome. Printouts from the Web site included a list of NCI active program announcements (PA), recently published RFAs, and recently cleared concepts for RFAs and PAs. Dr. Shen also distributed a summary about the Institute's Cancer Chromosomal Aberration Project (CCAP) and provided a Web address (http://www.nlm.nih.gov) for a hypertext link to a special issue of Nature Genetics containing data on recurrent human chromosomal abnormalities in human cancer.

Dr. Mishoe discussed an RFA on stem cell transplantation to establish alpha chimerism, which builds on previous studies at NHLBI. Applications for this RFA are due November 24, 1998. Dr. Mishoe also distributed a summary entitled "Concepts for FY99, 2000 and Beyond" that gives a brief overview of future research areas, but does not include budgetary amounts. NHLBI is preparing to readvertise the Stem Cell Biology Score Program, which should be announced in the spring of 1999.

Dr. Badman summarized NIDDK plans for an RFA on iron overload and iron overload therapies with a receive date in June, 1999. It is hoped that the RFA will be published in January 1999. This RFA arises from NIDDK's iron chelator program and is intended to take advantage of the new findings on regulation of iron transport and other recent studies. The Institute is ready to commit about $2 million in 2000 money. Five grants totaling $4.7 million a year were awarded recently for zebrafish genomic research using funds contributed by 13 Institutes and Centers. The NIDDK is the administering institute for these grants, which are for mapping the fish's genome and may lead to identification and cloning of the genes of interest. A trans-NIH program announcement for functional studies using the zebrafish has been released, and another announcement for additional research may be released later this year. A zebrafish meeting to be held at NIH on May 10-11 will focus on the developing genetic tools for the zebrafish. Among other NIDDK plans is a program announcement for research emphasizing protein and protein interactions in red cell membranes.

Dr. Badman, in closing the meeting, asked members to think about topics for future meetings. The next Hematology Subcommittee meeting will likely be after January 1, 1999. In addition, plans for information exchange at the American Society of Hematology December 4-9, 1998, were discussed. The contact for distribution of documents at the hematology display table is Grace Shen.

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