2000 Global Researcher Conference Proceeding
March 10 - 12, 2000
| Conference: | 2000 Global Researcher Conference |
|---|---|
| Title: | Toward gene therapy for nephrogenic diabetes insipidus |
| Authors: | Green, Lindsay; koeberl, D. D.; Filanoski, Brian J.; Cogdell, David E.; Wildin, Robert |
| Institutions: | Oregon Health Sciences University, (formerly) Oregon Health Sciences University, MP350, Duke University Medical Center |
The objective of this work is to develop gene therapy for genetic diseases of the renal tubule and collecting duct. The primary disease model is X-linked nephrogenic diabetes insipidus (NDI). Adeno-associated virus (AAV) is a non-immunogenic human parvovirus that is currently of interest as a potential gene therapy vector for many systems. The AVPR-2 gene mutated in NDI, and its promoter, are small enough to fit into an AAV vector genome. To determine whether AAV can infect renal tubule cells, AAV expressing an alkaline phosphatase (AP) reporter gene driven by the Moloney murine leukemia virus retroviral LTR promoter was used to infect two mouse renal tubule cell lines. Transduction of these cells was done in the presence or absence of helper [E1-]adenovirus (AV) to determine if the helper AV would enhance AAV transduction of cells. Two days after infection, cells were fixed and stained for AP. For the mouse inner medullary collecting duct cells (IMCD), 7.5 X 102 AP+ cells per mL of AAV vector alone, and 2.6 X 104 AP+ cells per mL of AAV vector were observed in the presence of helper AV. For the mouse distal convoluted tubule cells (DCT), no AP+ cells were found for AAV vector alone, and 1.9 X 104 AP+ cells per mL of AAV were observed in the presence of helper AV. Chromosomal integration of the AAV genome, which contains a neomycin phosphotransferase gene, in transduced cells was shown by selection for G418 resistant colonies. Two days after infection, G418 was added (at 0.75 mg/ml) and selection continued until colonies formed and all cells in uninfected wells had died. IMCD cells formed 3.25 X 103 colonies per mL of AAV vector in the presence of helper AV. Based upon our observations, we conclude that AAV is suitable for infecting mouse renal tubule cells and has a reasonable probability for long-term expression of transduced genes. We also conclude that the presence of the helper adenoviral genes enhances AAV infection of the mouse renal tubule cells.
To ensure that AVPR-2 genes delivered by AAV in vivo are expressed only where the endogenous gene is active, 5' flanking sequences that function as a promoter in cell culture were defined. In transient transfection experiments, a 2 kb segment of 5' flanking sequence plus intron A drives expression of reporter genes (EGFP and n-ßgal) in several renal cell lines including DCT, IMCD and MDCK. The same segment driving EGFP appeared to cause unilateral renal medullary death in one chimeric transgenic mouse, who was otherwise healthy. We conclude that this AVPR-2 promoter has the potential to restrict vectored gene expression to the appropriate cell type. Together, these results support the feasibility of gene therapy for NDI and other diseases of the distal tubule and collecting duct using AAV.,/p>
An essential part of any gene therapy is discovering or developing the best transportation medium (vector) by which the therapeutic agent is delivered to its target in the human body. Often this vector is a virus. Green, et al., tested the adeno-associated virus (AAV) to see if it would be a suitable vector for genetic therapies directed at genetic diseases of the kidney tubules and collecting ducts such as NDI.
They infected two mouse kidney cell lines by transferring an alkaline phosphatase (AP) reporter gene into the cell lines via AAV. Some of the AAV vectors contained a helper adenovirus (AV) to see if it would enhance AAV's effectiveness as a vector. Two days after they infected the cells, the researchers examined them to see if the AP had been successfully transferred to the cell lines. Based on their findings, the researchers concluded that AV is suitable for infecting mouse renal tubule cells and that the transferred genes, once in their new host, have a reasonable probability of being able to express themselves over time.