Transgene expression in polarized cells

Details Transgene expression in polarized cells Transgene expression in polarized cells

1999-07-01

2002-10-15

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C514S002600, C514S04400A, C424S093100, C424S093200, C424S093600, C435S455000, C435S456000, C435S325000, C435S320100

Reexamination Certificate

active

06465007

ABSTRACT:

The present invention relates to the novel use of compounds that disrupt tight junctions to facilitate the intracellular delivery (and/or transfection) of biologically active molecules by lipids, cationic amphiphilic compounds, non-viral and viral vectors.
The effective introduction of foreign genes and other biologically active molecules into targeted mammalian cells is a challenge still facing those skilled in the art. Gene therapy requires successful transfection of target cells in a patient. Transfection, which is practically useful per se, may generally be defined as a process of introducing an expressible polynucleotide (for example a gene, a cDNA, or an mRNA) into a cell. Successful expression of the encoding polynucleotide thus transfected leads to production in the cells of a normal protein and is also practically useful per se. A goal, of course, is to obtain expression sufficient to lead to correction of the disease state associated with the abnormal gene.
Examples of diseases that are targets of gene therapy include: inherited disorders such as cystic fibrosis, Gaucher's disease, Fabry's disease, and muscular dystrophy. Representative of acquired target disorders are: (1) for cancers—multiple myeloma, leukemias, melanomas, ovarian carcinoma and small cell lung cancer; (2) for cardiovascular conditions—progressive heart failure, restenosis, and hemophilias; and (3) for neurological conditions—traumatic brain injury.
Cystic fibrosis, a common lethal genetic disorder, is a particular example of a disease that is a target for gene therapy. The disease is caused by the presence of one or more mutations in the gene that encodes a protein known as cystic fibrosis transmembrane conductance regulator (“CFTR”). Cystic fibrosis is characterized by chronic sputum production, recurrent infections and lung destruction (Boat, T. F., McGraw-Hill, Inc., 1989, p. 2649-2680). Though it is not precisely known how the mutation of the CFTR gene leads to the clinical manifestation (Welsh, M. J. et al.
Cell
73:1251-1254, 1993), defective Cl secretion and increased Na
+
absorption (Welsh, M. J. et al.,
Cell
73:1251-1254, 1993; Quinton, P. M.,
FASEB Lett.
4:2709-2717,1990) are well documented. Furthermore, these changes in ion transport produce alterations in fluid transport across surface and gland epithelia (Jiang, C. et al.,
Science
262:424-427, 1993; Jiang, C. et al.,
J. Physiol.
(
London
), 501.3:637-647, 1997; Smith, J. J. et al.
J. Clin. Invest,
91:1148-1153, 1993; and Zhang, Y. et al.,
Am.J.Physiol
270:C1326-1335, 1996). These resultant alterations in water and salt content of airway liquid (ASL) may diminish the activity of bactericidal peptides secreted from the epithelial cells (Smith, J. J. et al.,
Cell,
85:229-236, 1996) and/or impair mucociliary clearance, thereby promoting recurrent lung infection and inflammation.
It is widely expected that gene therapy will provide a long lasting and predictable form of therapy for certain disease states such as CF, however, there is a need to develop improved methods that facilitate entry of functional genes into cells, and whose activity in this regard is sufficient to provide for in vivo delivery of genes or other such biologically active molecules.
Effective introduction of many types of biologically active molecules has been difficult and not all the methods that have been developed are able to effectuate efficient delivery of adequate amounts of the desired molecules into the targeted cells. The complex structure, behavior, and environment presented by an intact tissue that is targeted for intracellular delivery of biologically active molecules often interfere substantially with such delivery. Numerous methods and delivery vehicles including viral vectors, DNA encapsulated in liposomes, lipid delivery vehicles, and naked DNA have been employed to effectuate the delivery of DNA into the cells of mammals. To date, delivery of DNA in vitro, ex vivo, and in vivo has been demonstrated using many of the aforementioned methods.
Though viral transfection is relatively efficient, the host immune response frequently poses a major problem. Specifically, viral proteins may activate cytotoxicity T lymphocytes (CTLs) which destroy the virus-infected cells thereby terminating gene expression in the lungs of in vivo models examined. The other problem is diminished gene transfer upon repeated administration of viral vectors due to the development of antiviral neutralizing antibodies. These issues are presently being addressed by modifying both the vectors and the host immune system, however, a more efficient method of viral transfection or delivery is also desirable.
For example, the relatively low efficacy of AdV mediated gene transfer to airway epithelial cells is a major barrier for gene therapy of CF. Gene therapy with recombinant adenoviral (AdV) vectors may also lead to inflammatory and immune responses. For applications in which repeat therapy is necessary, such as CF, these responses can limit the therapeutic usefulness of the vector. In principle, the utility of vectors may be improved by increasing its therapeutic index, i.e., by either increasing its efficacy or decreasing its toxicity. For example, a strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given amount of transgene expression, and thereby also reduce unwanted effects such as immune responses.
Gene transfer using AdV has been proposed as a method to treat CF. Although the ability of AdV vectors to deliver the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells has been demonstrated by many laboratories, this process has been shown to be relatively inefficient. In addition, while treatment with AdV expressing CFTR has been shown to correct the chloride channel defect in human CIF airway epithelia grown in culture, the ability to correct the enhanced sodium absorption exhibited by these cells has been much less apparent. As a result of this inefficiency, relatively high doses of AdV need to be administered in vivo to observe significant correction of physiologic deficits. Due to the undesirable host immune response associated with delivering high doses of AdV, it is desirable that both the transfer efficiency and level of expression from AdV be improved to develop an effective treatment for CF.
Additionally, non-viral and non-proteinaceous vectors have been gaining attention as alternative approaches. Because compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both non-polar and polar environments (in or on, for example, the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself, such compounds are designed typically to contain both polar and non-polar domains. Compounds having both such domains may be termed amphiphiles, and many lipids and synthetic lipids that have been disclosed for use in facilitating such intracellular delivery (whether for in vitro or in vivo application) meet this definition. One group of amphiphilic compounds that have showed particular promise for efficient delivery of biologically active molecules are cationic amphiphiles. Cationic amphiphiles have polar groups that are capable of being positively charged at or around physiological pH, and this property is understood in the art to be important in defining how the amphiphiles interact with the many types of biologically active molecules including, for example, negatively charged polynucleotides such as DNA.
Several recently issued U.S. patents, the disclosures of which are specifically incorporated by reference herein, have described the utility of cationic amphiphiles to deliver polynucleotides to mammalian cells. (U.S. Pat. No. 5,676,954 to Brigham et al. and U.S. Pat. No. 5,703,055 to Felgner et al.)
Although the compounds mentioned in the above-identified references have been demonstrated to facilitate the entry of biologically active

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