ILAR Journal Online, Volume 36(3/4) 1994: Advances in Gene Therapy

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ILAR Journal V36(3/4) 1994 [FORMERLY ILAR NEWS]
Advances in Gene Therapy

Abstracts from the Michigan State University Genetics Program Symposium on Advances in Gene Therapy

Saturday, September 10, 1994, Lansing, Michigan

Dystrophin Expression Vectors for Gene Therapy of Muscular Dystrophy

Jeffrey S. Chamberlain, Ph.D.
Department of Human Genetics
University of Michigan Medical School
Ann Arbor, Michigan

My laboratory is exploring the feasibility of gene therapy for Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy and one of the most common human genetic diseases. We have created a variety of dystrophin expression vectors that are being analyzed in mdx mice, a mouse model for DMD. As dystrophin is too large for most viral delivery systems, we have created a variety of dystrophin vectors deleted for various domains of the protein and are asking whether these clones can lead to an elimination of dystrophic symptoms in mice. Various levels of wild-type dystrophin have been produced in transgenic mice, which provide data on the minimal levels of the protein needed to eliminate symptoms. Those studies are being complemented by analysis of the truncated constructs. The ability to eliminate dystrophic symptoms by overexpression of truncated cDNAs will facilitate the use of viral vectors for gene therapy of DMD.

In a parallel series of studies we are developing adenovirus vectors for delivery of the truncated clones. Initially we are testing a variety of muscle-specific enhancer plus promoter elements in an attempt to create muscle-specific adenoviruses that express transgenes at high levels. These vectors are being created with reporter genes and with the truncated dystrophin clones as the gene to be expressed. These studies are ongoing. An update of progress was presented.

Viral Vectors for Hepatic Gene Therapy

Mark A. Kay, M.D., Ph.D.
Markey Molecular Medicine Center
Division of Medical Genetics
University of Washington
Seattle, Washington

Our goal is to develop hepatic gene transfer as a means for treating hemophilia B. We have pursued three approaches in the mouse and factor IX deficient canine animal models. The first is the ex vivo approach which involves surgical removal of 20% of the liver, dissociation of hepatocytes for cell culture, trb. nsduction of therapeutic genes with recombinant retroviral vectors, and finally transplantation of the hepatocytes back into the liver. To develop these methods in normal dogs we used a retroviral vector that expresses the human a-l-antitrypsin (hAAT) cDNA. This protein was quantitated in the serum of recipients by an immunologic assay. After developing the methods to transplant 5% to 10% of the original hepatocyte mass back into the animal, the hAAT vector was used to transduce the hepatocytes prior to transplantation. We were able to demonstrate up to 4.6 ng/ml of serum hAAT in recipients for about 1 month. The transduced cells remained functional in the liver, however, loss of gene expression was due to shutoff of the CMV promoter that was used in the retroviral construct. The ex vivo method is labor intensive, thus, we developed a relatively simple method for introducing genes into the hepatocytes of animals by direct infusion of virus after partial hepatectomy. Because the retrovirus requires cell division for gene transfer, we performed a two-thirds hepatectomy in mice to stimulate cell division followed by the infusion of different retroviral vectors. Using the intracellular b-galactosidase marker, we were able to demonstrate that after gene transfer about 1% to 2% of the hepatocytes express genes in vivo. After performing similar experiments with retroviral vectors that express hAAT, serum hAAT persisted in the serum of recipients for the life of the animal. This method allowed us to test a larger number of vectors for gene expression from the liver in vivo. We adapted this method to the hemophilia B dog model and used a retrovirus that expressed the factor IX cDNA. After gene transfer, the animals had a reduction in their whole blood clotting time from about 50 minutes to 20 minutes. The plasma factor IX concentrations were in the range of 2 to 10 ng/ml (0.1% of normal concentrations) as determined by a biological assay and immunologic assay. Additionally, there was a significant reduction in the partial thromboplastin times (PTTs) after therapy. Although this represents partial correction of hemostasis, an important finding was that expression is constitutive and has continued to persist for one year. The third approach uses recombinant adenoviral vectors. In contrast to the retrovirus, these vectors will infect non-dividing cells, thus, there is no need to perform partial hepatectomy. Gene transfer into hepatocytes results in transduction of almost 100% of cells in the mouse and at least 50% in the dog. We constructed vectors that express canine factor IX which when transferred into the livers of deficient animals resulted in more than 3 times the normal concentration of factor IX. The animals were tested and found to have normal PTTs, whole blood clotting times, and bleeding times. Expression was transient however, and no factor IX was present in the plasma of recipients 80 to 100 days after treatment. The transient expression was due to antigen-specific immunologic response directed against transduced hepatocytes.

The retroviral-mediated gene transfer into hepatocytes results in permanent and long-term low-level gene expression. Before clinical application, we believe that gene expression will need to be increased by 50- to 100-fold. Adenoviral-mediated hepatic gene transfer results in high levels of gene expression albeit expression is transient. For adenovirus to be useful for hemophilia gene therapy, expression will need to be more persistent or there will need to be a method to readminister the virus in a noninvasive manner.

Advances in Fragile X Syndrome: Genetic and BiochemicalAspects

Stephen T. Warren, Ph.D.
William Patterson Timmie Professor of Human Genetics
Departments of Biochemistry and of Pediatrics
Emory University School of Medicine
Howard Hughes Medical Institute
Atlanta, Georgia

Fragile X syndrome is an X-linked dominant disorder with reduced penetrance that accounts for a substantial proportion of mental deficiency in humans. Fully penetrant males exhibit moderate mental retardation, macroorchidism (enlarged testes), subtle facial dysmorphia, and mild connective-tissue abnormalities. Female patients are typically less severely affected, showing little or no somatic signs and only borderline to mild mental retardation. The gene responsible for fragile X syndrome, FMR1, was identified in 1991 by Dr. Warren and an international group of collaborators.

The molecular basis of fragile X syndrome is attributed to the expansion of an unstable CGG trinucleotide repeat in the 5' untranslated region ofFMR1. This repeat is normally polymorphic (mode of 30 triplets) and genetically stable but displays remarkable instability in fragile X families. In affected individuals, the repeat is expanded beyond ~200 trinucleotides, usually to (CGG)_700-1,000. Nonpenetrant carriers of either sex typically have alleles with 60-200 triplets, and only the female alleles expand beyond 200 repeats when transmitted. Fragile X syndrome was the first example of this novel trinucleotide repeat mutation, which was subsequently found to be responsible for seven other genetic diseases, including myotonic dystrophy and Huntington's disease.

In fragile X syndrome, when the size of the CGG repeat is beyond 200 triplets, the FMRI gene is concomitantly methylated, resulting in transcriptional silencing. The absence of FMR1 message and its encoded protein, FMRP, results in the phenotype of fragile X syndrome. The normal function of this protein, whose absence leads to mental retardation, is under investigation in Dr. Warren's laboratory as is the mechanism that leads to trinucleotide repeat instability and expansion.

Just Caring: Ethical Issues in Germ-Line Genetic Therapy

Leonard M. Fleck, Ph.D.
Center for Ethics and Humanities in the Life Sciences
Michigan State University
East Lansing, Michigan

We start with the assumption that there are no strong moral objections in principle to somatic cell genetic engineering. This is because it is morally analogous to other experimental medical therapies. But human germ-line genetic engineering seems radically different, in part, because the "patients" are four-celled embryos and their descendants. That is, these are individuals who cannot give free and informed consent for what will be done to them. Moreover, it is not clear that there are any morally appropriate surrogate decisionmakers.

The strong claim has been made by some that human germ-line genetic engineering is seriously morally objectionable; and therefore, it is a medical technology that ought neither be developed nor deployed. In this essay I consider six arguments ih support of this claim.

In brief, the six objections I consider are as follows: (1) Germ-line genetic engineering violates altogether the moral autonomy of the future individuals so engineered. (2) Germ-line genetic engineering involves "playing God." It involves excessive human hubris. We lack the wisdom to make changes that are potentially so profound and far-reaching. In addition, what we are changing is the human genetic patrimony, something that none of us has an individual right to tamper with. (3) Germ-line engineering is excessively risky, both morally and medically. We can achieve the same therapeutic goals through morally less objectionable means, such as somatic cell engineering or embryo selection after genetic analysis. (4) Germ-line engineering involves excessive embryo destruction. There is a moral inconsistency in this practice because we pretend to have a therapeutic attitude toward the embryo as patient while at the same time maiming and destroying most of the embryos treated. (5) Given the likely very high costs associated with germ-line genetic engineering, and given the likelihood that only those already well off would likely be able to afford it and given the vast unmet health needs of the poor and uninsured, justice requires not developing or deploying germ-line genetic engineering. (6) There is a morally "slippery slope" from respect for reproductive freedom of individuals who would like to see their future children protected from dread genetic disorders to eugenics, which would involve socially mandated genetic engineering of children. The only way to avoid this moral slide is to forbid the development of the technology from the beginning.

In the final analysis I find all of these objections less than compelling because there are competing moral considerations that weaken their moral force. I conclude that germ-line genetic engineering should go forward, though we may discover as the technology unfolds that there are specific applications of it that are morally objectionable.