Research Portfolio

Funding Opportunities

Join our Mailing List
Join our mailing list to be notified of new funding opportunities.

Your Email

To receive information about funding opportunities, events, and program updates.



Gene therapy for emphysema

Institution: University of California, San Francisco
Investigator(s): Francis Szoka, Ph.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6RT-0109 Award: $443,179
Subject Area: Pulmonary Disease
Award Type: Research Project Awards
Abstracts

Initial Award Abstract
Gene therapy offers great promise as a new class of drugs; i.e., a treatment that can correct hereditary or environmentally-caused diseases in humans. A therapeutic gene can be delivered into a cell by viral and non-viral methods, and this gene will direct the cell to produce its own specific type of medicine. Viral methods can efficiently deliver genes to cells in animals, however, associated safety issues have dampened the initial enthusiasm for using viruses in the treatment of humans. Cationic liposomes, the most efficient non-viral gene delivery vector, are microscopic lipid (fat-based) vesicles which bind to DNA and facilitate cellular uptake.

We are developing liposome molecules to carry genes into cells of the lung to help combat diseases such as emphysema, which is the consequence of low levels of the protein alpha-1-antitrypsin (AT). Our objective is to understand how these fat-based molecules mediate gene delivery to the lungs of experimental animals. We will deliver the tagged liposome-DNA complexes to mice either by administration into the lung or by intravenous injection, and study the fate of each component, the liposome and DNA, in the lung over a time course. We will also deliver complex mixtures marked with red (liposome) and green (DNA) tags, and visually identify the cell type in the lung with the attached tag, and then follow the fate of both liposomes and DNA within the cell under the microscope. We will then correlate these results with the levels of human AT protein produced in the mouse. We will also alter the properties of the liposome-DNA complex by co-adding agents which rapidly free DNA from the complex. In addition, we can add different molecules to the liposome-DNA complex which cause them to bind to specific cell types in the lung, or cause enhanced cellular uptake of the liposome-DNA complex. These studies will potentially lead to the development of a new class of drugs which can be used to treat tobacco-related diseases such as emphysema.

Final Report
Gene therapy offers great promise as a new class of drugs; a medicine that can correct hereditary diseases in humans. A therapeutic gene can be delivered into a cell by viral and non-viral methods, and this gene will direct the cell to produce its own specific type of medicine. We are developing liposomes to carry genes into cells of the lung to help combat diseases such as emphysema which is the consequence of expression of low level of the protein alpha-1-antitrypsin (AT). Our objective is to understand how these lipid molecules mediate gene delivery to animals. We will deliver the tagged liposome-DNA complexes to mice either by instillation into the lung or by intravenous injection, and study the fate of each component, the liposome and DNA, in the lung over a time course. We will also deliver complexes marked with red (liposome) and green (DNA) tags, and microscopically identify the cell type in the lung with the attached tag, and then follow the fate of both liposomes and DNA within the cell. We will then correlate these results with the levels of human AT protein produced in the mouse. We will also try to alter the properties of the liposome-DNA complex by co-adding agents which rapidly free DNA from the complex. In addition, we can add different molecules to the liposome-DNA complex which cause them to bind to specific cell types such as macrophages or alveolar type II cells in the lung, or causes enhanced cellular uptake of the liposome-DNA complex.

During the three years of the research we have studied the mechanism of delivery using cationic liposomes using two routes of administration into mice: through the blood and though the airways. The results of our research show that changes of the liposome formulations, by including an additional lipid result in substantially improved gene expression in the lung. We further show that the alpha-1-antitrypsin protein is expressed in the distal regions of the lung after the gene is delivered in these lipoplexes precisely where lung damage occurs during emphysema. We have discovered that gene delivery occurs in the first 60 minutes after lipoplex I.V. administration , that plasma proteins such as albumin, complement, fibrinogen, lipoproteins as well as platelets are not important factors in gene delivery. However, cell surface heparin is a major factor regulating gene delivery. These studies have been accepted for publication. Thus we have improved gene expression in the lung by two orders of magnitude, have shown expression of alpha-1-antitrypsin in the lung by two routes of administration, have developed a biodegradable lipid and we have improved our understanding of the time course and mechanism of transfection. We are have also examined gene delivery via the airways and using the two color confocal microscopic method have shown that gene delivery occurs only in areas that receive both the lipid and the DNA from the lipoplex. We further show that the major reason lipoplex mediated DNA delivery via the airways is less efficient than delivery via the blood is that there are regions of the lung that do not come in contact with the delivery system. Thus intratracheal administration results in poor distribution of the dose throughout the lung. This suggests that if the dose was given by aerosolization into the lungs there would be improved distribution and higher levels of gene expression.

We have also developed novel methods to improve DNA transfection by using cationic detergents that self-assemble on the DNA backbone and form a crosslinked lipid. Using this technique we have assembled nanometric complexes with less then a 50 nm diameter. These complexes should be more efficient at entering cells via the endosome and therefore may lead to improved gene delivery. We are now testing whether or not the nanometric complexes improve gene therapy to the lung. A major finding of these studies is that toxicity from cationic lipoplexes can be reduced by reducing the cationic component. These are significant findings for the grant period and set the stage for further improvements for gene therapy of emphysema using improved gene carriers.
Publications

Effects of complement on the pharmacokinetics and gene delivery mediated by cationic lipid-DNA complexes
Periodical: Human Gene Therapy Index Medicus:
Authors: Barron LG, Meyer KB, Szoka FC Jr ART
Yr: 1998 Vol: 9 Nbr: Abs: Pg: 315-323

Synthesis and characterization of long chain alkyl acyl carnitine esters. Potentially biodegradable cationic lipids for use in gene delivery
Periodical: Journal of Medicinal Chemistry Index Medicus:
Authors: Wang J, Guo X, Xu Y, Barron L, Szoka FC Jr ART
Yr: 1998 Vol: 41 Nbr: Abs: Pg: 2207-2215

Biochemical, morphological and functional analysis of a cyclic peptide, phospholipid and DNA tenary complex used for gene therapy
Periodical: Journal of Liposome Research Index Medicus:
Authors: Legendre JY, Huang S-K, Szoka FC Jr ART
Yr: 1998 Vol: 8 Nbr: Abs: Pg: 347-366

Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes
Periodical: Human Gene Therapy Index Medicus:
Authors: Plank C, Tang MX, Wolfe AR, Szoka FC Jr. ART
Yr: 1999 Vol: 10 Nbr: Abs: Pg: 319-332

Cationic lipids are essential for gene delivery mediated by intravenous administration of lipoplexes
Periodical: Human Gene Therapy Index Medicus:
Authors: Barron LG, Uyechi LS, Szoka FC Jr. ART
Yr: 1999 Vol: 6 Nbr: Abs: Pg: 1179-1183

Lipoplex-mediated gene delivery to the lung occurs within 60 minutes of intravenous administration
Periodical: Human Gene Therapy Index Medicus:
Authors: Barron LG, Gagne L, Szoka FC Jr. ART
Yr: 1999 Vol: 10 Nbr: Abs: Pg: 1683-1894

Physicochemical characterization and purification of cationic lipoplexes
Periodical: Biophysical Journal Index Medicus:
Authors: Xu Y,Hui S-W, Frederik P, Szoka FC ART
Yr: 1999 Vol: 77 Nbr: Abs: Pg: 341-353

Controlled template-assisted assembly of plasmid DNA into nanometric particles with high DNA concentration
Periodical: Bioconjugate Chemistry Index Medicus:
Authors: Ouyang M, Remy J-S, Szoka FC ART
Yr: 2000 Vol: 11 Nbr: 1 Abs: Pg: 104-112

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
Periodical: Human Gene Therapy Index Medicus:
Authors: Uyechi LS, Thurston G, Gagne L, Szoka Jr FC ART
Yr: 2001 Vol: 8 Nbr: Abs: Pg: 828-836