MadSci Network: Genetics
Query:

Re: How do they get the CF gene into the adenovirus in Gene Therapy?

Area: Genetics
Posted By: Sharon Shriver, Instructor (faculty; Ph.D.), Dept. of Pharmacology (I do molecular genetics), University of Pittsburgh
Date: Tue Sep 2 10:03:35 1997
Area of science: Genetics
ID: 871525196.Ge
Message:

Dear Wayne,

Gene therapy is one of the most interesting and valuable techniques to come out of the field of genetic engineering. The adenovirus used to deliver the CF gene to human cells for gene therapy is known as a vector. Since most cells normally will not take in or absorb DNA, we need a delivery system to get the gene into the cell. Adenovirus is a good vector since it can infect cells in vivo, or while they’re in the body, which means that the gene can be delivered through an inhaler (instead of having to manipulate the cells in vitro, or in the laboratory, then return the cells to the body). To use the adenovirus as a vector, it’s genome was first altered by removing all the virus DNA except for the minimum necessary for the virus to live and infect the cells. Genetically engineered viral vectors like this are harmless and usually can’t live outside of the laboratory.

A clone, or copy, of the CF gene was then inserted into the viral genome, or what was left of it, so that the virus thinks the CF gene is part of it’s own DNA. We can “cut and paste” DNA in this way using enzymes which have been purified from other viruses and bacteria. Restriction enzymes cut (or restrict) DNA at specific DNA sequences, and ligases attach free DNA ends together. In this way the unwanted genes are cut out of the viral genome and the CF gene is pasted in. The viral genome would then be called recombinant. We then use other purified proteins and enzymes to build a new virus, or “package” the DNA. To do this, the recombinant DNA is mixed with all of the protein parts of the virus (for the outer coat, and the parts that attach to the cell, for example) along with some viral enzymes that assemble the virus, and in the test tube a whole, intact virus is created with the new DNA inside. After infecting some cells in the laboratory, the new virus makes millions of copies of itself (carrying the CF gene), which can be purified and used for gene therapy.


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