SFHR, An Alternative to Classic Gene Therapy

Spring 1998

by Dr. Dieter C. Gruenert, Ph.D., Associate Professor, Associate Director of the Gene Therapy Core Center at the University of California in San Francisco, and Genetics Advisor for IACFA

Editor's Note: The prospect of directly correcting the mutant portion of disease-causing genes is one of the main goals of gene therapy. In the following article, Dr. Gruenert discusses his work with a technique called Small Fragment Homologous Replacement (SFHR), an alternative to classic gene therapy approaches, which he believes will target only the mutant portion of the cellular DNA. CFRI has funded Dr. Gruenert several times since 1986. In the spring of 1997, CFRI awarded a one-year grant to Dr. Gruenert to conduct tests using SFHR.

My work over the last 12 years at the Gene Therapy Core Center at the University of California in San Francisco has been almost exclusively directed toward human airway epithelial cell and molecular biology as it relates to cystic fibrosis (CF). With initial funding from CFRI and the Cystic Fibrosis Foundation (CFF), we were able to be the first laboratory to establish immortal cell lines (giving cells the potential to live indefinitely) from normal human and CF airway epithelial cells. Our work eventually led to the establishment and characterization of surface airway and submucosal epithelial cell lines of cystic fibrosis patients. Our laboratory provides these cell lines to laboratories throughout the world to aid in the understanding of the physiological and genetic mechanisms that underlie CF and other airway diseases, including asthma and even cancer.

In addition to being critical for enhancing the understanding of the basic defect that underlies cystic fibrosis, the cells have been important in the development of new therapies for treating CF. In particular, these cells have played a critical role in the development of novel gene therapy strategies for CF. We have focused our efforts on developing an approach to gene therapy that involves directly correcting the mutated region of the CF gene with wild-type (normal) CF Transmembrane Conductance Regulator (CFTR) gene sequences. This technique is called Small Fragment Homologous Replacement (SFHR). This method is significant because it replaces "bad" genetic material with "good" in the CF patient without destroying the structure of the CFTR gene or altering the regulatory elements. While this strategy for gene therapy is not yet ready for the clinic, we are investigating the mechanisms underlying homologous replacement using both in vitro (outside the body) and in vivo (in the living body) model systems.

Efforts have been carried out within my laboratory as well as with a collaborator, Dr. William Colledge, of Cambridge University in the United Kingdom, to determine how feasible it is to use SFHR for in vivo correction of the CF ion transport. Our initial studies were carried out in normal mice. In these mice, the airways were exposed to a fragment containing mutant (DF508 CFTR) sequences to determine if the DNA fragments would actually get to the cells expressing CFTR and ultimately be expressed themselves as messenger RNA. The results of these studies indicated that the DF508 sequences present in the fragment were, in fact, expressed as messenger RNA, implying that replacement had occurred in cells that normally express CFTR. This finding suggests it may be possible to reach and correct CFTR mutations in cells that naturally express CFTR in people with cystic fibrosis.

Further work is now under way to evaluate the potential for SFHR to correct the chloride ion transport defect in the nasal mucosa of CF mice homozygous for (that carry two copies of) the DF508 mutation. While the results for these studies are in the preliminary phase, the data suggests that chloride ion transport can be restored. To verify this finding, we will undertake numerous additional studies to evaluate how many applications of SFHR are necessary to correct the chloride transport defect, and to determine how long this correction will last.

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