Endocrine Abstracts

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of tumours of the parathyroids, pancreas and pituitary. MEN1 is caused by germline inactivating mutations of the MEN1 gene which is located on chromosome 11q13 and encodes a 610 amino acid protein, menin. MEN1 tumours show loss of heterozygosity (LOH) of chromosome 11q13, and lack menin expression, consistent with a tumour suppressor role for MEN1. Hence MEN1 gene replacement therapy is a potential treatment. We therefore aimed to develop an adenoviral gene therapy vector capable of delivering the wild-type murine Men1 gene to menin-deficient tumours arising in Men1 (+/−) knockout mice. An adenovirus infectivity test was performed on pituitary tumour cell cultures, obtained from Men1+/− mice, using a recombinant Green Fluorescence Protein (GFP)-expressing adenovirus. GFP expression was detected over a wide range of multiplicities of infection, thereby demonstrating that tumour cells from Men1+/− mice could be transduced by a serotype 5 adenovirus (Ad5). An expression cassette, that contained the murine Men1 cDNA under the control of the cytomegalovirus (CMV) promoter, was assembled into a transfer plasmid through multiple cloning steps. Transient expression of this plasmid in human embryonic kidney (HEK-293) cells showed that it was able to express menin. The expression cassette was subsequently transferred to an E1/E3 deleted Ad5 genome, which renders the virus replication-deficient, by homologous recombination in E. coli. HEK-293 cells transfected with the recombinant adenoviral DNA produced viral plaques. Viral particles were further amplified and purified, and Western blot analysis demonstrated that the resulting recombinant adenovirus was capable of expressing menin in vitro.

Thus, we have established a recombinant adenoviral vector capable of expressing menin in a mammalian cell system, which can be of use for gene therapy studies of MEN1 tumours.