Experiment 1. Gene transfer of hFIX into hemophilia B mice:

The rAAV vectors were transfected into the hepatocytes of normal mice, via intra-portal infusion. Southern Analysis was performed to determine the distribution of the rAAV genome, over a 13-week period, in various tissues from these mice.

What properties should a vector have for long term gene therapy? Which would you choose?

• Vector should be able to lyse cells?
• Vector should integrate without gene expression?
• Vector should integrate into host genome with vector expression?
• Vector should be able to replicate and produce functional progeny?

What are adeno-associated viruses, (AAV)?

Adeno-associated viruses are non-pathogenic human viruses. They are members of the Parvovirus family and are icosahedrally symmetrical and un-enveloped. The Parvovirus genome is made up of linear, monopartite ssDNA.that are dependant on a helper virus, (usually adenovirus), to proliferate. The AAV single stranded DNA genome contains two genes, rep and cap, which code for the required replicating and capsid proteins. Adeno-assoceated viruses contain within their promotor sequence a control element called the rep binding site, (rbs). AAV produces initial rep proteins that bind to the rbs causing minimal expression of the other rep proteins neccessary for reproduction of this virus. The helper virus interferes with the binding of the proteins at the rep site, allowing for expression of the rep proteins. In the absence of a helper virus, AAVs integrate into a specific point in the host genome with a high frequency. Integration occurs through a recombination event. This ability to integrate into the host genome makes AAV a good choice for a vector to confer long-term gene expression.

 

 What is the rAAV vector?

The adeno-associated viral vector was constructed by digesting AAV nucleic acid sequences with the restriction endonuclease, XbaI to remove almost the entire genome, leaving only the terminal repeats. The following elements were inserted between the terminal repeats; MFG promotor, the Moloney murine leukemia virus (MuLV)5' long terminal repeat, adjacent splice donor/acceptor sequences, the canine factor IX cDNA sequence, (cFIX), and the poly(A) site of bovine growth hormone,(bGH pA). Two other vectors used have the same components but cFIX was replaced with hFIX (human factor IX) cDNA, or hTH (human tyrosine hydroxylase) cDNA, which will be used as a control. The following is an illustration of the three recombinant vectors used in this study:

What is the functional significance of the components in the rAAV construct?

• MFG promotor region is from the MFG retrovirus, which has been successfully used in other similar studies. Their reason for including the MFG sequence is that it contains a splice site where an intron can be inserted. It has been demonstrated that inclusion of an intron increases gene expression, invivo. Therefore inclusion of the MFG promotor is for enhancement of gene expression.
• The MuLV is a 5'LTR region from a transducing retrovirus called Murine Leukemia Virus. Most of the retroviral vectors used are based on the Moloney Murine Leukemia Virus backbone. Then additional retroviral regions can be inserted to modify and improve gene expression or increase transduction effeciency.
• The adjacent splice donor/acceptor sequence allows for insertion of the gene of interest.
• The bGH pA is a termination sequence allowing for termination of transcription of the gene.

 To view the results of this experiment, click here